Nucleic Acids Symposium Series - recent issues

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2009-09-12

Prelims

2009-09-12

Reaction of the 4'-benzenesulfonyl derivatives of thymidine with organosilicon and organoaluminum reagents

Shimada, H., Kikuchi, S., Okuda, S., Haraguchi, K., Tanaka, H. — 2009-09-12

With an aim to develop a new approach to synthesize 4'-substituted nucleosides, reactions of thymidine derivatives having a benzenesulfonyl leaving group at the 4'-position with organosilicon and organoaluminum reagents were investigated. Two substrates 4 (-L-isomer) and 4 β (β-D-isomer) were prepared for this purpose. Although reaction of 4 with organosilicon reagents gave preferentially the 4'-substituted (allyl and N₃) β-D-nucleoside, its reaction with AlMe₃ gave the 4'-methyl--L-thymidine as the major product. On the other hand, the substrate 4β, upon reacting with AlMe₃, furnished the desired 4'-methylthymidine exclusively in high yield.

Alternative I-D exchange reaction on pyrimidine and purine nuclei mediated by tributyltin hydride using THF-d8 as a deuterium source

Mutsumi, T., Maruhashi, K., Monguchi, Y., Sajiki, H. — 2009-09-12

A novel method for the regioselective deuteration of pyrimidine and purine rings mediated by Bu₃SnH using THF-d₈ as a deuterium source on the basis of a radical reaction was developed.

Rational design and synthesis of ansa-adenosines as potential antitumor agents

Muranaka, K., Ichikawa, S., Matsuda, A. — 2009-09-12

Synthesis of benzoquinone ansa-adenosines, which are rationally designed as Hsp90 inhibitors by extracting and fusing a natural substrate, ATP, and a natural product, geldanamycin, was described. This simpler scaffold design provides practical synthesis of a set of analogs and demonstrates synthetic innovation.

Development of next generation 4'-selenonucleosides

Jeong, L. S., Tosh, D. K., Choi, W. J. — 2009-09-12

Next generation 4'-selenonucleosides as potential antiviral and antitumor agents were synthesized using a Pummerer type condensation as a key step.

Stereocontrolled synthesis of oligodeoxyribonucleoside boranophosphates via stereodefined H-phosphonate intermediates

Iwamoto, N., Oka, N., Wada, T. — 2009-09-12

P-Stereodefined oligodeoxyribonucleoside boranophosphates (PB-ODNs) were succecfully synthesized by the conversion of diastereopure H-phosphonate intermediates. Thermal denaturation studies clearly showed that the duplex stability of PB-ODNs with the complementary DNA and RNA depends on the stereochemistry of the boranophosphate linkages.

Synthesis and properties of a novel 2',4'-BNA bearing a urea bridged structure

Yahara, A., Nishida, M., Baba, T., Kodama, T., Imanishi, T., Obika, S. — 2009-09-12

Three kinds of novel bridged nucleic acid (BNA) monomers bearing carbamate or urea bridged structures, were designed and synthesized. One of these monomers, a urea-type BNA monomer was successfully incorporated into oligodeoxynucleotides (ODNs), and properties of the ODNs were evaluated. The ODNs containing the urea-type BNA monomers showed RNA selective hybridizing profiles and significant enzymatic stability.

Synthesis and properties of terminally modified oligonucleotides capable of short-RNA selective hybridization

2009-09-12

We have developed 2'-O-methyl-RNAs having phosphorylated cyclohexane at the 5' and/or 3'-terminal adenine. These 2'-O-methyl-RNAs formed less stable duplex with the longer target RNA than that with the short target RNA. We tried to improve the short-RNA selective hybridization property by the synthesis of 2'-O-methyl-RNAs having terminal guanine modifications.

Biochemical dissection of RISC assembly and function

Tomari, Y. — 2009-09-12

Small silencing RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), regulate expression of their target genes via RNA-induced silencing complex (RISC). RISC assembly follows complex, ordered pathways, and RISC function is as diverse as cleavage, translational repression, and deadenylation of the target. We have recently shown how siRNAs and miRNAs are assembled into distinct types of RISC, and how differently they function, using Drosophila as a model organism.

Metal ion binding of modified pyrimidine-base pairs in DNA duplexes

Ono, A., Iwamoto, K., Sugiyama, K., Okamoto, I. — 2009-09-12

We report the synthesis and metal ion-binding properties of DNA duplexes containing 5-substituted uracil (^XU) pairs, such as 5-bromouracil, 5-fluorouracil and 5-cyanouracil pairs. Thermal denaturation studies of these modified DNA duplexes revealed that the DNA duplexes were stabilized in the presence of Hg^II ions in acidic and neutral solutions. On the other hand, the duplexes were stabilized in the presence of Hg^II and Ag^I ions. These results indicated that ^XU-Hg^II-^XU complex was formed in the acidic and neutral solutions, then, in the basic solutions ^XU-Ag^I-^XU complex as well as the ^XU-Hg^II-^XU complex were formed. ESI-TOF MS analysis also indicated formation of the metal ion-DNA complexes.

Interaction of silver ion with CG.C+ base triplets in DNA triplex

Ihara, T., Ishii, T., Jyo, A. — 2009-09-12

When designing ligands for specific sequences in DNA duplexes, triple helix formation is a useful recognition motif, because base triplet formation is based on the simple rule of complementary Hoogsteen hydrogen bonding, CG.C⁺ and TA.T. However the triplexes containing CG.C⁺ triplets form only in a weak acidic solution, because cytosines in third strand need to be protonated to satisfy its complementarity to CG base-pairs. A simple and easy method to stabilize the DNA triplex using Ag⁺ was reported. A silver ion displaces the N3 proton of cytosine in Hoogsteen base-pairing to form a base triplet, CG.CAg⁺. By the addition of an equimolar amount of Ag⁺, the third strand 15 mer sequence containing five cytosines was stabilized by ca. 30°C in melting temperature at pH 7. The triplex structure was stable even under weak basic conditions.

Synthesis and triplex formation of oligonucleotides containing 8-thioxodeoxyadenosine and 5-methyl-2-thiodeoxycytosine

Ohkubo, A., Miyata, K., Tsunoda, H., Seio, K., Sekine, M. — 2009-09-12

For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides (TFO) containing 8-thioxodeoxyadenine (s⁸A) residues in place of the protonated cytosines (Cs) required for the third base pairing with DNA duplexes. Consequently, it was found that s⁸A exhibited much stronger hybridization ability than C under neutral conditions when four s⁸A bases were arranged in a consecutive sequence. Moreover, we also synthesized TFOs containing 5-methyl-2-thiocytosines and examined their ability of triplex formation.

Development of a novel device to trap heavy metal cations: Application of the specific interaction between heavy metal cation and mismatch DNA base pair

Torigoe, H., Miyakawa, Y., Fukushi, M., Ono, A., Kozasa, T. — 2009-09-12

We have already found that Hg(II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4 °C. We have also found that Ag(I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4 °C. Using the specific interaction, we developed a novel device to trap each of Hg(II) and Ag(I) cation. The device is composed of 5'-biotinylated T-rich or C-rich DNA oligonucleotides, BIO-T20: 5'-Bio-T₂₀-3' or BIO-C20: 5'-Bio-C₂₀-3' (Bio is a biotin), immobilized on streptavidin-coated polystylene beads. When the BIO-T20-immobilized beads were added to a solution containing Hg(II) cation, and the beads trapping Hg(II) cation were collected by centrifugation, almost all of Hg(II) cation were removed from the solution. Also, when the BIO-C20-immobilized beads were added to a solution containing Ag(I) cation, and the beads trapping Ag(I) cation were collected by centrifugation, almost all of Ag(I) cation were removed from the solution. We conclude that, using the novel device developed in this study, Hg(II) and Ag(I) cation can be effectively removed from the solution.

Role of microRNAs in the pathogenesis of human cance

Croce, C. M. — 2009-09-12

Over the past few years, molecular oncology research has revealed that abnormalities in both protein coding genes (PCGs) and noncoding RNAs (ncRNAs) can be identified in tumors and that the interplay between PCGs and ncRNAs is causally involved in the initiation, progression and metastases of human cancers. MicroRNAs (miRNAs), which are among the most studied ncRNAs, are small 19- to 25-nucleotide genes involved in the regulation of PCGs and other ncRNAs. With the recent findings of miRNAs' involvement in cancer, miRNAs are strongly associated with the pathogenesis in human cancers. In this review, we focus on the possible mechanisms of miRNAs in cancer pathogenesis.

Synthesis and properties of oligodeoxynucleotides containing biaryl units

Ueno, Y., Komatsuzaki, S., Takasu, K., Kitamura, Y., Kitade, Y. — 2009-09-12

We describe the synthesis and properties of oligonucleotides (ONs) containing biaryl units, which are composed of a bis(hydroxymethyl)benzene residue and a naphthalene or pyrene moiety. We found that by introducing the biaryl units into the ONs, the aromatic chromophores are suitably arrayed in the DNAs. Further, we succeeded in the detection of a single-base mismatch in RNA by using the ON containing the biaryl unit as a molecular beacon.

Efficient energy transfer from pyrene to perylene assembled inside DNA duplex

Kashida, H., Takatsu, T., Asanuma, H. — 2009-09-12

Pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to increase the apparent Stokes’ shift of perylene. Multiple donors were introduced in the vicinity of acceptors via D-threoninol and natural base pairs were inserted between donors and acceptors in order to suppress undesired interactions between them. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 495 nm was observed from perylene when excited at 345 nm where pyrene has its absorption. The apparent Stokes’ shift became as large as 115 nm with a high FRET efficiency (>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R₀) of the donor pyrene.

Investigation of the mechanism of the fluorescent emission of the silylated pyrene modified DNA

Moriguchi, T., Hattori, M., Sekiguchi, T., Shinozuka, K. — 2009-09-12

Modified DNA containing silylated pyrene derivative at the 5'-terminus showed the obvious discrimination ability of the duplex formation, that the modified DNA showed the strong fluorescence emission in the presence of the complementary DNA, in spite of the very weak emission in the absence of the complementary DNA. The intensity of the fluorescence emission of the double strand found to increase about 10 times compared with that of the single strand. In this paper, we tried to disclose the detailed mechanism of such a discrimination ability of the silylated pyrene-modified DNA.

Toward a reciprocal evolution system between RNA and peptides as an artificial model for the early RNP world

Ikawa, Y., Furuta, H., Yamashita, K., Kashiwagi, N. — 2009-09-12

In the early stages of the evolution of life, RNA–polypeptide complexes (RNPs) have been suggested to play crucial roles. At a certain developmental stage of ancient RNPs, their RNA and polypeptide components could evolve in an interdependent manner to develop complex structures and functions. To mimic this possible process, we have designed an RNA molecule that can act as a template for chemical peptide ligation. This designed RNA possesses two peptide-binding sites that capture the two basic peptides. The designed RNA actually facilitated the peptide ligation. The resulting ligated peptide, which has two RNA binding sites, can in turn function as a trans-activator that enhances the intrinsic ribozymatic activity of the designed RNA.

Substrate recognition of pre-tRNA by ribonuclease P --- subsite model of natural ribozyme originated from Escherichia coli

Fujimoto, A., Kikuchi, Y., Tanaka, T. — 2009-09-12

Combination of cleavage site analysis and kinetic analysis of a series of shape variant of pre-tRNA substrate, we newly found two subsites which contribute to recognition of shape in substrate binding and catalysis by bacterial RNase P. Our data showed that the ribozyme traps the substrate by 5'- and 3'-end regions to form Michaelis complex, and after that the shape of the substrat is examied by other two subsites in the transition state. In the meeting, we will show the posibility that the S-domain can contribute to stabilize the transition state of the cleavage reaction of a pre-tRNA substrate.

The NEXT-A (N-Terminal EXtension with Transferase and ARS) reaction

Taki, M., Kuroiwa, H., Sisido, M. — 2009-09-12

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA^Phe by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides A294G mutation on the ARS, T251A, βG318W, or βA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

The exceptional properties of Plasmodium deoxyguanylate pathways as a potential area for metabolic and drug discovery studies

Kandeel, M., Kitamura, Y., Kitade, Y. — 2009-09-12

In Plasmodium falciparum, deoxyguanylate was found to be a substrate for several DNA metabolizing enzymes. Guanylate kinase utilizes dGMP with very low specificity, which is estimated to be the lowest among well-known prokaryotic and eukaryotic enzymes. Furthermore, thymidylate kinase, which is a pyrimidine specific enzyme, was found to phosphorylate dGMP with a surprisingly high specificity similar to that of the natural substrate. The above mentioned distinctions are specific for the Plasmodium protozoa and provide an interesting method for tracking dGMP metabolism during development and a starting point for drug development.

Structural basis for the specificity of thymidylate kinases from human pathogens: implications for nucleotide analogues activation

Meyer, P., Caillat, C., Topalis, D., Balzarini, J., Deville-Bonne, D. — 2009-09-12

Several human pathogens possess nucleoside or nucleotide kinases with large substrate specificity compared to their human counterparts. This phenomenon has been successfully exploited for the specific targeting of prodrugs such as Acyclovir against herpes virus. Combined structural and biochemical studies of these enzymes can thus provide essential information for the rational design of specific antimicrobial agents. Here we studied the structural basis for the specificity of a thymidylate kinase from the poxvirus family. Poxvirus thymidylate kinase has unusual substrate specificity and can accept bulky analogues such as 5-bromo-vinyl-dUMP (BVdUMP). The 2 Å crystal structure of the thymidylate kinase bound to this compound now gives the structural basis for its specific molecular recognition.

Chemical methods to study protein-nucleic acid interactions

He, C. — 2009-09-12

Accumulation of genetic changes due to the presence of unrepaired DNA lesions can lead to cancer development and other diseases. Nucleic acid modifications also play key roles in many essential life processes. We have developed a series of chemically modified nucleic acid analogues that can be applied to stabilize protein-nucleic acid interactions for structural and proteomic studies. Some of the probes have also been employed to study nucleic acid-nucleic acid interactions.

Development of novel chemical probes to detect abasic sites in DNA

Kojima, N., Takebayashi, T., Mikami, A., Ohtsuka, E., Komatsu, Y. — 2009-09-12

We chemically synthesized a series of aminooxy derivatives to develop novel probes for sensitive detection of abasic (AP) sites in DNA. The results of the conjugation reactions showed that the probes could efficiently react to AP sites by introducing an aromatic or a guanidino group in their structures. In particular, the probe having both functional groups showed the most effective reactivity, indicating that hydrophobic and electrostatic interactions cooperatively acted in the reaction of the probe to AP sites. We then synthesized a biotinylated probe and succeeded in more sensitive detection of AP sites in genomic DNA than with the conventional aldehyde reactive probe (ARP).

Assessment of the DNA damage using the fluorescence microscope

Yamazaki, Y., Zinchenko, A., Murata, S. — 2009-09-12

DNA damage was monitored by fluorescent microscopy observations of DNA fluorescent images after hydrodynamic stretching on a microscope glass. DNA double-strand breaks lead to a decrease of the average length of observed fluorescent DNA molecules. Compared to conventional methods such as electrophoresis, the proposed method allows for the analysis of the DNA damage at very low DNA breaking frequency. In particular, this method was used to study DNA damage by weak UV irradiation in solutions of quantum dots.

Exciton-controlled fluorescence: Application to hybridization-sensitive fluorescent DNA probe

Okamoto, A., Ikeda, S., Kubota, T., Yuki, M., Yanagisawa, H. — 2009-09-12

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Multiple activities of c-di-GMP in Pseudomonas aeruginosa

Lory, S., Merighi, M., Hyodo, M. — 2009-09-12

Survival strategies of many bacterial pathogens, including Pseudomonas aeruginosa, are linked to their ability to form surface associated communities called biofilms. The biofilm life style allows these organisms to persist in various tissues, avoid clearance by innate host defences and significantly enhanced their resistance to antibiotics. Formation of various biofilm components, including the synthesis of the extracellular polysaccharide matrix, is controlled at the transcriptional and translational levels and also by a small molecule second messenger bis-(3',5')-cyclic-di-guanidine monophosphate (c-di-GMP). The synthesis of c-di-GMP from GTP and its degradation is controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), encoded by over thirty genes in the P. aeruginosa genome. We have shown that an increase in the intracellular c-di-GMP levels favors biofilm formation due to its role as a cofactor for the synthesis of several types of extracellular polysaccharides, including PEL and alginate, the two key virulence factors of P. aeruginosa during infection of patients with cystic fibrosis. During biosynthesis of PEL and alginate, c-di-GMP binds to specific receptors, PelD and Alg44, respectively. We have also recently demonstrated that DGCs have a relaxed specificity and can cyclize other nucleotides besides GTP. These atypical cyclic dinucleotides bind c-di-GMP receptors with high affinity, suggesting that intracellular regulation of various biological functions by this group of second messengers may be more complex than previously recognized.

Development of peptide-oligonucleotide conjugates for regulation of small RNA function

Yamayoshi, A., Momokawa, D., Kobori, A., Murakami, A. — 2009-09-12

Recently, various microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) have been identified and play important roles in gene regulatory networks. However it is a little known about their biological functions. These small RNAs exhibit their function to form a ribonucleoprotein complex, the RISC (RNA-induced silencing complex), which modulates gene expression by translational repression. In this study, we developed a novel peptide antagonist to inhibit the RISC function. The peptide was conjugated to 2’-O-methyl oligoribonucleotides, which have a complementary sequence to the guide strand of siRNA, and regulatory effects of the peptide on RISC activity were examined. It was revealed that the peptide drastically enhanced inhibitory effects of the oligonucleotide on RISC activity. Here we demonstrate our peptide-oligonucleotide conjugate that can provide a powerful and specific way to regulate the small RNA function.

Studies on the effects of arginine residues introduced to peptide ribonucleic acids (PRNA) on the complex stability with RNA

2009-09-12

In this study, a series of novel -Peptide ribonucleic acid (-PRNA) oligomers, possessing alternative -PRNA/arginine or serine sequences, were newly designed, synthesized, and evaluated as the third-generation PRNA. As expected, these -PRNAs formed highly stable sequence-specific complexes with the complementary RNAs, for which both the conventional hydrogen-bonding interactions between the complementary nucleobase pairs and the electrostatic interactions between the arginine's guanidinium cation and the RNA's phosphate anion on the backbone are jointly responsible. Moreover, in the cases of -PRNA and single point mismatched DNA mixing systems, appreciable T_m could not be observed, thus -PRNAs containing Arg were expected to have high nucleobase sequence discrimination abilities. It was demonstrated that the recognition behavior of -PRNA with Arg/Ser backbone with complementary RNA can be controlled externally through the orientation change of pyrimidine nucleobase induced by borate ester formation of the ribose's 2',3'-cis-diol.

Polyethylenimine derived nanoparticles for efficient gene delivery

Pathak, A., Patnaik, S., Gupta, K.C. — 2009-09-12

Introduction of therapeutic genes into the cells of an organism in a safe and efficient way has become a challenging task in non-viral mediated gene therapy. Here, branched polyethylenimine (bPEI, 25 kDa) was converted into nanoparticles through electrostatic interactions with anionic polysaccharides (e.g. alginic acid, Al and hyaluronic acid, HA). A small library of PEI-Al and PEI-HA nanoparticles was synthesized by varying the amounts of anionic polysaccharides and evaluated in terms of their size, surface charge, cytotoxicity, transfection efficiency, etc. Both the series of nanoparticles exhibited higher cell viability and transfection efficiency as compared to native PEI and the standard transfection reagents. In vivo targeting efficacy of PEI-HA(4.6%) nanoparticles was examined in tumor induced mice.

DNA junction structure stabilized by molecular crowding conditions

Miyoshi, D., Muhuri, S., Mimura, K., Sugimoto, N. — 2009-09-12

We examined the effects of molecular crowding conditions on the structures and thermodynamics of three-way junctions (TWJs) of DNA. To explore the crowding effects on the junction point, we further evaluated the number of water molecules associated with the whole TWJ as well as the individual arms. It was found that the number of water molecules taken up by the whole TWJ was significantly smaller than the sum of the individual arms. These results clearly show the dehydration from the junction point of the TWJ structure. Therefore, molecular crowding should be favourable for the junction point of TWJ structure and unfavourable for the duplex structure. From these results, it can be concluded that a cell-mimicking molecular crowding condition in which the activity of water decreases and hydration becomes less favourable, might facilitate the formation of junction structures in comparison with duplexes.

Cationic comb-type copolymer as a nucleic acid chaperone for DNA quadruplex

Moriyama, R., Shimada, N., Kano, A., Maruyama, A. — 2009-09-12

The intermolecular DNA quadruplex is attractive materials for design of nanomolecular constructs and machines. This folding kinetics is, however, slow, hampering its application to these materials. We have reported that the cationic comb-type copolymer accelerated duplex and triplex formation. In this study, the effect of the copolymer on the intermolecular quadruplex folding was investigated. The copolymer was found to accelerate the association of intermolecular quadruplex by three-orders. Furthermore, the copolymer induced the strand exchange reaction between intermolecular quadruplex and its single-stranded counterparts. We suggested that the copolymer acts as a nucleic acid chaperone for the intermolecular quadruplex.

Molecular basis of human telomere DNA/RNA structure and its potential application

Xu, Y., Suzuki, Y., Kaminaga, K., Komiyama, M. — 2009-09-12

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. However, structure and function of the telomeric RNA in chromosome ends have not yet been elucidated. Using a combination of NMR, CD and MALDI-TOFMS experiments, we have demonstrated that human telomere RNA can form a parallel G-quadruplex structure. Furthermore, we successfully found for the first time that human telomere DNA and RNA sequence can form a DNA-RNA hybrid type G-quadruplex structure based on click chemistry approach. Telomerase or its telomere DNA substrate is also known to present a specific target in discovering anticancer agents. Recently, we developed a structure-based approach to sequence-specific cleaving of human telomeric DNA by G-quadruplex formation. These results not only provide valuable information to allow understanding of the roles of human telomeric RNA in telomere biology, but also serve as a start step for developing new anti-cancer reagent.

Synthetic nanocircular RNA for controlling of gene expression

Abe, H., Abe, N., Uda, M., Tsuneda, S., Ito, Y. — 2009-09-12

We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops¹. RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme, and are thus transformed into double-stranded RNA in cells, although this RNA is resistant to degradation in serum. The structure was optimized to maximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA.

Site-specific modification of RNA by functionality-transfer ODN probes

Onizuka, K., Taniguchi, Y., Nishioka, T., Sasaki, S. — 2009-09-12

Efficient methods for the modification of RNA molecules have been expected as innovative biological tools and therapeutic methods. In this study, the development of a general method for site-specific RNA modification by the functionality-transfer ODN probes has been investigated. Site-specific and cytosine-selective RNA modifications were achieved by the functionality-transfer reaction. It was shown that the base and site-selectivity were due to the close proximity of the reactants in the DNA-RNA duplexes.

Molecular design of sequence specific DNA alkylating agents

Minoshima, M., Bando, T., Shinohara, K.-i., Sugiyama, H. — 2009-09-12

Sequence-specific DNA alkylating agents have great interest for novel approach to cancer chemotherapy. We designed the conjugates between pyrrole (Py)-imidazole (Im) polyamides and DNA alkylating chlorambucil moiety possessing at different positions. The sequence-specific DNA alkylation by conjugates was investigated by using high-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The results showed that polyamide chlorambucil conjugates alkylate DNA at flanking adenines in recognition sequences of Py-Im polyamides, however, the reactivities and alkylation sites were influenced by the positions of conjugation. In addition, we synthesized conjugate between Py-Im polyamide and another alkylating agent, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). DNA alkylation reactivies by both alkylating polyamides were almost comparable. In contrast, cytotoxicities against cell lines differed greatly. These comparative studies would promote development of appropriate sequence-specific DNA alkylating polyamides against specific cancer cells.

Stable DNA triple helix formation

Fox, K. R., Cardew, A. S., Vergara, N., Brown, T. — 2009-09-12

Triplex-forming oligonucleotides bind in the major groove of duplex DNA, generating three stranded structures containing T.AT and C⁺.GC triplets. Their sequence specific binding has potential uses in gene targeting but is limited by their low affinity, the requirement for low pH and the need for oligopurine targets. We have prepared nucleotide analogues to overcome these limitations and can now target some sequences that contain pyrimidine interruptions at physiological pH. We have tested the biological activity of these modified oligonucleotides. TFOs that contain multiple substitutions with positively charged groups bind with high affinity and we have explored how they interact with secondary sites.

Site-specific incorporation of functional components into RNA by transcription using unnatural base pair systems

Kimoto, M., Sato, A., Kawai, R., Yokoyama, S., Hirao, I. — 2009-09-12

The creation of an extra, unnatural base pair that functions in replication, transcription, and translation, would provide a new system for the expansion of the genetic alphabet. In transcription, an unnatural base pair system could be used for making new RNA molecules containing functional components of interest at specific positions. We have developed several unnatural base pairs that can function in replication and transcription. Among them, the 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) pair and the Ds and pyrrole-2-carbaldehyde (Pa) pair exhibit high selectivity and efficiency in PCR amplification and T7 transcription, respectively. We performed PCR amplification involving the Ds-Px pair for DNA template preparation and T7 transcription involving the Ds-Pa pair with several modified Pa substrates for the incorporation of functional components, such as biotin- and fluorescein-linked Pa nucleotides, into RNA. This Ds-Px and Ds-Pa pair system could provide a powerful tool for the site-specific fluorescent labeling and immobilization of RNA molecules, as a detection system for RNA and RNA-protein interactions.

Restriction enzyme treatment/ligation independent cloning using caged primers for PCR

Kuzuya, A., Tanaka, K., Katada, H., Komiyama, M. — 2009-09-12

We report here a new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment. By using caged primers in PCR, unnatural sticky-ends of any length and sequence are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in desired arrangement, tightly enough to be repaired and ligated in competent cells. We have successfully constructed a recombinant vector based on pBR322 and coding GFP gene by applying this simple and effective system.

Kinetic studies of long-range hole transfer through DNA

Kawai, K., Majima, T. — 2009-09-12

We report the kinetic studies of hole transfer through DNA based on the transient absorption measurements of photosensitizer naphthalimide (NI) and hole acceptor phenothiazine (PTZ) modified DNA.

Effective algorithm to encrypt information based on self-assembly of DNA tiles

Hirabayashi, M., Kojima, H., Oiwa, K. — 2009-09-12

We present an error-tolerance scheme to encrypt information in DNA structures based on a one-time-pad (OTP) cryptosystem that provides theoretically unbreakable security. The problem of the DNA-based OTP encryption is the loss of synchronization between the message and the encryption key due to the DNA property of accepting mismatched base pairs. We propose a new implementation idea of encrypting algorithm with the fourfold fault tolerance against mismatches than the ordinary DNA XOR operation. Although there are several problems to be solved toward the practical use at this moment, it is expected that the molecular computation using DNA tiles will enlarge the application possibility of the OTP cryptosystem.

Three-dimensional DNA nanostructures constructed by folding of multiple rectangles

Endo, M., Sugiyama, H. — 2009-09-12

The novel multi-arm DNA structures were designed using 2D-DNA Origami method, and these structures were folded into 3D hollow prism structures by introduction of connection strands into the arms. The opening of the prism structures were examined by a high-speed AFM, which showed the dissociation events of the connecting arms in the 3D-structures.

Defining the syntax for self-assembling RNA tertiary architectures

Jaeger, L. — 2009-09-12

Stable RNAs are modular and hierarchical three-dimensional (3D) architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions (1, 2). Using comparative sequence and structural analysis of known X-ray structures of RNAs, folding and assembly principles of RNA can presently be gathered to generate the syntax of a proto-language for rational design and prediction of RNA 3D shapes. RNA architectonics refers to the deciphering of this proto-language and to its use to build new functional RNA shapes with self-assembly properties (3-5). This approach can therefore contribute to the prediction and rational design of RNA tertiary structures for potential applications in nanotechnology, synthetic biology and medicine.

One-electron oxidation of DNA: mechanism and consequences

Schuster, G. B. — 2009-09-12

All living organisms store the information necessary to maintain life in their DNA. Any process that damages DNA and causes loss or corruption of that information threatens the viability of the organism. One-electron oxidation is such a process. Loss of an electron from DNA generates a radical cation that is located primarily on its nucleobases. The radical cation migrates reversibly through duplex DNA by hopping until it is eventually trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In its normal aqueous solutions, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counter ions (typically Na⁺) to the phosphate groups play an important role in facilitating both the migration of the radical cation and in its eventual reaction with H₂O. Irreversible reaction of a radical cation with H₂O in duplex DNA occurs preferentially at the most reactive site. In normal DNA that is comprised of the four common DNA nucleobases, reaction occurs most commonly at a guanine and results in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step primarily by a tandem process. The general outcomes revealed in the one-electron oxidation of DNA oligomers in solution appear to be generally valid also for more complex DNA structures and for the cellular DNA of living organisms.

Structure and real-time monitoring of the enzymatic reaction of APOBEC3G which is involved in anti-HIV activity

2009-09-12

Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) is known to play a role in intrinsic cellular immunity against human immunodeficiency virus type 1 (HIV-1). The antiretroviral activity of APOBEC3G is associated with hypermutation of viral DNA through cytidine deamination. APOBEC3G contains two cytidine deaminase domains that are characterized by a highly conserved zinc-coordinating motif. It is known that only the C-terminal domain of APOBEC3G (c-APOBEC3G) is involved in the catalytic activity. Here, we present the solution structure and the interaction with single-stranded DNA of c-APOBEC3G. Furthermore, we have succeeded for the first time in monitoring the deamination reaction of c-APOBEC3G in real-time using NMR signals. The monitoring has demonstrated that the deamination reaction occurs in a strict 3'->5'

NMR studies of DNA recognition mechanism of HMGB1 protein

Furuita, K., Murata, S., Jee, J., Ichikawa, S., Matsuda, A., Kojima, C. — 2009-09-12

A 2'-deoxyuridylate dimer cyclized via cross-linkage by an ethylene (U^et_pU) or a propylene (U^pr_pU) linker at the 5-position was incorporated into DNA oligomers. Fluorescence resonance energy transfer (FRET) experiments showed that they bent at approximately 90°. We investigated binding abilities of U^et_pU and U^pr_pU DNA oligomers to HMGB1 A-box protein, which specifically binds to bent DNA, using nuclear magnetic resonance (NMR) spectroscopy. Both DNA oligomers bind to HMGB1 A-box protein, however, the U^et_pU DNA oligomer has higher affinity than the U^pr_pU DNA oligomer. In order to explain this difference, we studied the solution structures of the U^et_pU and U^pr_pU DNA oligomers using NMR. Most ¹H signals except for 4', 5' and 5'' were assigned. Cross-peak patterns of ¹H-¹H NOESY spectra indicate that both oligomers have right-handed B-form like structures and the cyclization in 2'-deoxyuridylates does not break Watson-Crick base pairs. Chemical shift differences between these two DNA oligomers suggest the presence of the local structural differences in the region of 2'-deoxyuridylate dimer and its 3' side between the U^et_pU and U^pr_pU DNA oligomers.

Analysis of the spacial requirements for RNA-protein interactions within the N antitermination complex of bacteriophage {lambda}

2009-09-12

In bacteriophage , formation of a transcriptional antitermination complex consisting of the N protein, nut RNA transcript (boxA-boxB), host factors, and RNA polymerase is mediated by the interaction of the boxB RNA with the RNA-binding domain of N. In order to understand the spacial requirements of this boxB/N interaction within the complex, the effects of changes in the length of the nut site linker, the boxB stem, and the peptide spacer connecting the RNA-binding domain and activation domain of N were examined using a bacterial reporter system. As a result, we found that the requirements for the boxB stem length and N peptide linker length were optimized and strict. In contrast, when the boxB/N interaction was replaced by heterologous RNA/peptide interactions, the strict requirement for the length of the peptide linker and the RNA stem was relaxed, presumably due to the absence of the interaction between boxB/N and the host factor NusA in the wild-type complex. It was also shown that the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer, suggesting that a further understanding of the organization of the antitermination complex may provide insights into the engineering of functional ribonucleoprotein complexes.

Monitoring biological interactions using perforated evanescent-field-coupled waveguide-mode nanobiosensors

2009-09-12

Evanescent-field-coupled (EFC) waveguide-mode sensors recently been shown to be suitable for detecting various biomolecules. In the present studies, we demonstrated that both nucleic acids hybridization and nucleic acids-protein interactions can be analyzed using perforated evanescent-field-coupled waveguide-mode nanobio-sensors.

9-(2-C-Cyano-2-deoxy-{beta}-D-arabino-pentofuranosyl)guanine, a potential antitumor agent against B-lymphoma infected with kaposi's sarcoma-associated herpesvirus

2009-09-12

Several 9-(2-C-cyano-2-deoxy-l-β-D-arabino-pentofuranosyl)purine derivatives were tested against Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cells. The guanine derivative (2, CNDAG) as well as the 2-amino-6-substituted-purine derivatives 3, 4 and 5 inhibited KSHV-positive cell growth but showed no cytotoxicity against KSHV-negative cells at >15 µM concentrations. Therefore, it was found that compounds 2, 3, 4 and 5 showed selective cytotoxicity against PEL cells infected with KSHV.

Synthesis and anti-HIV activity of 4'-C-ethynyl-2'-deoxy-4'-thio-nucleosides

2009-09-12

Synthesis of 4'-C-ethynyl-2'-deoxy-4'-thionucleosides was carried out based on electrophilic glycosidation using 4-C-ethynyl-4-thiofuranoid glycal. The glycal 15 was prepared as follows: oxidative cleavage of 6 with Pb(OAc)₄ forming the aldehyde 7, aldol reaction of 7 and subsequent silylation to furnish 8, conversion of the formyl group of 8 into an ethynyl group, and finally β-elimination of the resulting 14 with t-BuLi. The glycosyl donor 16 was prepared by silyl-protection of 15. Electrophilic glycosidation was performed between silylated N⁴-acetylcytosine and 16 in the presence of N-iodosuccinimide. Radical-mediated removal of the introduced iodine atom followed by deprotection gave 4'-C-ethynyl-2'-deoxy-4'-thiocytidine (18).

Synthesis of 4'-substituted cordycepins via benzenesulfenylation at the 4'-position as a key step

Kubota, Y., Ehara, M., Kumamoto, H., Shimada, H., Haraguchi, K., Tanaka, H. — 2009-09-12

With an aim to synthesize 4'-substituted cordycepins, the 4'-phenylthio precursor 4 was prepared from adenosine through an electrophilic addition to the 3',4'-unsaturated derivative 2 by using NIS/PhSH system. Nucleophilic substitution of 4 with a series of alcohols in the presence of NBS gave the respective 4'--alkoxy cordycepins 6 as the major stereoisomer. Use of DAST, in stead of alcohol in this reaction, gave the 4'-fluoro analogue 7. The 4'-sulfone derivative 8 obtained by m-CPBA oxidation of 4 was employed for the reaction with organoaluminum reagents. These reactions furnished various types of the 4'-carbon-substituted cordycepins 9.

Synthesis of 5'-fluoro-2'-{beta}-methylneplanocin analogues

Kumamoto, H., Kobayashi, M., Tanaka, H. — 2009-09-12

Synthesis of 5'-fluoro-2'-β-methylneplanocin analogues (5) was carried out. The cyclopentenone 16 was prepared from methyl mannopyranoside by using ring closing metathesis, stereoselective introduction of methyl group, and seleno cyclization as representative steps. Introduction of a fluorine atom was conducted by electrophilic fluorination. Antiviral activity of 5 will also be presented.

Synthesis and anti-HCV activity of 2',5'-deoxy-5'-phenacyladenosine analogs

Ikejiri, M., Ohshima, T., Fukushima, A., Shimotohno, K., Maruyama, T. — 2009-09-12

Several nucleoside analogs containing a methylene group instead of a 5'-O atom were synthesized to study the effect of the 5'-modification of nucleoside analogs on their anti-HCV activity. Among the analogs, a 5'-phenacyl analog exhibited good anti-HCV activity with an EC₅₀ of 15.1 µM. This compound is hypothesized to function via a novel type of mechanism that does not involve the conventional 5'-O-triphosphorylation process.

Synthesis of deuterated benzyladenine and its application as a surrogate

2009-09-12

Palladium on carbon-ethylenediamine complex [Pd/C(en)] catalyzed deuteration of N⁶-benzyladenine-d₅, which is a plant growth regulator, to introduce 5 deuterium atoms, while use of Pd/C as a catalyst led to a complete removal of N⁶-benzyl group. The corresponding deuterated N⁶-benzyladenine was successfully used as a surrogate compound for the quantitative analysis of residual benzyladenine in crops using LC/MS/MS

Synthesis and properties of a novel artificial nucleoside that acts as a redox switch

Baba, T., Kodama, T., Imanishi, T., Obika, S. — 2009-09-12

Oligonucleotide that can reversibly alter the duplex-forming ability in response to an external factor has potential to be applied to various genome technologies, e.g. antisense and gene diagnosis technology. A novel bridged nucleoside which is capable of sensing an oxidant and reductant was designed and synthesized. The nucleoside formed a disulfide bridge between C2' and C4' positions, which would enhance its duplex-forming ability under oxidative conditions. Contrastingly, the disulfide bridge was cleaved under reductive conditions; it could cause decrease of duplex-forming ability.

The use of conformationally rigid nucleoside probes to study the role of sugar pucker and nucleobase orientation in the thrombin binding aptamer

2009-09-12

Thrombin binding aptamers (TBAs) incorporating North-/South-deoxyguanosines built on the rigid bicyclo[3.1.0]hexane template were synthesized. Individual 2'-deoxyguanosines at positions dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. The substitution at position 14 with a locked South/syn-dG nucleoside produced an aptamer with the same stability and global structure as the innate, unmodified one. Replacing position 15 with the same South/syndG nucleoside induced a strong destabilization of the aptamer, while the antipodal North/anti-dG nucleoside was less destabilizing. Remarkably, the insertion of a North/anti-dG nucleoside at position 14, where both pseudosugar conformation and glycosyl torsion angle are opposite with respect to the native structure, led to the complete disruption of the G-tetraplex structure as detected by NMR and confirmed by extensive molecular dynamics simulations.

Chemical synthesis of nucleoside H-boranophosphonates and their application as precursors of P-modified nucleotide analogues

Oka, N., Higashida, R., Takayama, Y., Ando, K., Wada, T. — 2009-09-12

Nucleoside H-boranophosphonates were synthesized via the condensation reactions of appropriately protected nucleosides with monopyridinium H-boranophosphonate and subsequent deprotection under basic conditions. 3',5'-O-Protected dinucleoside H-boranophosphonates were also synthesized via the condensation of the corresponding 5'-O-protected nucleoside 3'-H-boranophosphonates with 3'-O-protected nucleosides. A study on the chemical properties of the resultant dinucleoside phosphate analogues revealed that the P–H group could be further modified under anhydrous basic conditions.

Stabilization of A-type nucleic acid duplexes by novel oligodiaminosaccharides

Iwata, R., Sudo, M., Nagafuji, K., Wada, T. — 2009-09-12

Novel oligodiaminosaccharides, -(1->4)-linked-2,6-diamino-2,6-dideoxy-D-glucopyranose oligomers, were designed and synthesized to bind to A-type nucleic acid duplexes, such as RNA duplexes. Using properly designed glycosyl donors and glycosyl acceptors, an -selective glycosylation was achieved. A chain elongation cycle was established and the oligodiaminosaccharides bearing the -glycoside bonds (1-4mer) were synthesised. Analyses of their interactions with oligonucleotide duplexes were performed by using CD spectrometry and UV melting experiments. These experiments revealed that the 3mer and 4mer were found to remarkably stabilize RNA-RNA and RNA-DNA duplexes with small structural changes.

Synthesis and hybridization properties of oligonucleotides having 4-N-(pyrrol-2-ylcarbonyl)deoxycytidine

Yamada, K., Tsunoda, H., Ohkubo, A., Seio, K., Sekine, M. — 2009-09-12

A new 4-N-acylated deoxycytidine derivative, 4-N-(1H-pyrrol-2-ylcarbonyl)deoxycytidine, was synthesized and found to be stable under rather basic conditions. Oligodeoxynucleotides (ODNs) incorporating this modified deoxynucleoside at various positions were successfully synthesized by using a pivaloyloxymethyl (POM) group for protection of the pyrrole residue. The POM group was removed by treatment with 1.5 M NaOMe/MeOH. ODNs containing modified deoxycytidines exhibited hybridization properties superior to those of the unmodified ODNs. We found the acylation of the cytosine base with an aromatic acyl-type substituent led to significant increase of the thermo stability of DNA duplexes. This is the first noteworthy observation in this kind of modification. The synthesis and hybridization properties of 4-N-(1H-pyrrol-2-ylcarbonyl)deoxycytidine derivatives will be also reported.

Synthesis and properties of new RNA molecules incorporating 2'-O-aryluridine derivatives

Oeda, Y., Iijima, Y., Tsunoda, H., Okubo, A., Seio, K., Sekine, M. — 2009-09-12

A variety of 2'-O-arylnucleosides with functional groups were synthesized by the microwave-assisted reaction of 2,2'-anhydrouridine with phenol derivatives. The 2'-O-arylnucleosides thus obtained were incorporated into 2'-O-Me RNA or DNA oligomers and their hybridization properties were studied by T_m measurements.

Synthesis of modified double stranded RNAs containing duplex regions between amide-linked RNA and RNA at both ends and enhanced nuclease resistance

Iwase, R., Kurokawa, R., Ueno, J. — 2009-09-12

Synthetic short-interfering RNA (siRNA) has been a powerful tool to control gene expression. Chemical modification of the 3'-overhang regions of siRNA with amide-linked RNAs improved the exonuclease resistance and was compatible with RNAi function in cultured cells. To improve endonuclease resistance of the siRNA having amide-linked RNA modification at the 3'-terminal regions, we designed and synthesized new modified double stranded RNAs (dsRNAs) the 3'-terminal regions of which were modified with amide-linked RNA moieties and formed duplex structures with the complementary unmodified RNA moieties. The modified dsRNAs showed remarkable increase of stability in cell culture medium containing 10% serum in comparison with the modified siRNAs having 3'-overhang regions of amide-linked RNAs. The result suggests that the resistance of siRNA against endonuclease as well as 3'-exonuclease in serum can be enhanced by the introduction of the structures of amide-linked RNA:unmodified RNA duplexes at both ends of siRNA.

5'-Lipophilic conjugates of oligonucleotides as components of cell delivery systems

Iglina, A. A., Meschaninova, M. I., Venyaminova, A. G. — 2009-09-12

The approach to the synthesis of 5'-lipophilic conjugates of functional nucleic acids based on a combination of H-phosphonate and phosphoramidite methods is presented.

Introduction of 8-methyladenosine into 2', 5'-oligoadenylate (2-5A) 2'-terminus induces dramatic shift in binding site of RNase L

Nagaoka, K., Kito, S., Kitamura, Y., Ueno, Y., Kitade, Y. — 2009-09-12

The 2',5'-oligoadenylate (2-5A) system is an interferon (IFN)-regulated RNA decay pathway that provides innate immunity against viral infections. The biological action of the 2-5A system is mediated by RNase L, an endoribonuclease that becomes enzymatically active after binding to 2-5A. It has been reported that the 5'-phosphoryl group of 2-5A is required for RNase L activation. However, we have found that 5'-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2'-terminus were more effective as an activator of RNase L than the parent 2-5A (p5'A2'p5'A2'p5'A2'p5'A2'). Introduction of 8-methyladenosine is thought to induce a dramatic shift in the binding site of RNase L.

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by DNA polymerases

Minakawa, N., Ogata, S., Takahashi, M., Matsuda, A. — 2009-09-12

We describe herein the results of (i) enzymatic recognition for imidazopyridopyrimidine (Im):naphthyridine (Na) base pairs and (ii) further primer extension reactions after the Im:Na base pairs by DNA polymerases. Among the base pairs examined, ImN^O:NaO^N base pair was rather selectively recognized by Klenow fragment exo^– {KF (exo^–)} as complementary base. However, this DNA polymerase did not catalyze primer extension reactions after the ImN^O:NaO^N base pair. Therefore, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo^–), seemed most promising for this purpose.

Evaluation of base recognition abilities of 2'-deoxynucleoside N-oxide derivatives by polymerase reactions

Tsunoda, H., Ohkubo, A., Seio, K., Sekine, M. — 2009-09-12

The main products obtained by oxidation of cytosine and adenine bases with hydrogen peroxide are cytosine and adenine N-oxide derivatives. There is a possibility that these N-oxide derivatives are mutagenic in genomic DNA like 8-oxoguanine or thymine glycol. Although the chemical synthesis and properties of 2'-deoxynucleoside N-oxide derivatives have been well established, little has been reported about the chemical and biochemical behavior of oligodeoxynucleotides (ODNs) containing these modified 2'-deoxynucleoside. In this study, we examined their base recognition ability by DNA polymerase reactions. It was found that these modified derivatives were incorporated into the 3'-terminal site of an ODN by DNA polymerase selecting accurately the complementary G or T base on a template ODN. In the incorporation reaction using template ODNs containing 2'-deoxynucleoside N-oxide bases, their N-oxide bases were selectively recognized by the complementary 5'-triphosphate (dGTP or dTTP).

Preparation of eukaryotic mRNA having differently methylated adenosine at the 5'-terminus and the effect of the methyl group in translation

Ishikawa, M., Murai, R., Hagiwara, H., Hoshino, T., Suyama, K. — 2009-09-12

Cap analogues having differently methylated adenosine at 2' and N6 position, m⁷G^5'pppApG which is existed in plant mRNA (plant type), m⁷G^5'pppAmpG (animal type), m⁷G^5'pppm⁶AmpG (mammalian type) and m⁷G^5'pppm⁶ApG (unnnatural type), were synthesized. In order to clarify the function of these methyl groups, luciferase mRNAs having differently methylated adenosine at the 5'-terminus, were successfully prepared by in vitro transcription using the synthesized cap anologues. As the preliminary results of in vitro translation with rabbit reticulocyte lysate and luciferase assay, luciferase mRNA having the mammalian type of cap structure, m⁷G^5'pppm⁶AmpG, was most efficiently translated. In the case of m⁷G^5'pppApG (plant type) efficiency of translation was lowest.

Synthesis and characterization of deoxyuridine triphosphates labeled with pyrene

Tanimizu, Y., Takada, T., Nakamura, M., Yamana, K. — 2009-09-12

Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.

Duplex formation of multiple pyrene-modified RNAs

Fukuda, M., Nakamura, M., Takada, T., Yamana, K. — 2009-09-12

We synthesized multiple pyrene-modified RNA sequences having two kinds of consecutive sequences, U_PyU_Py and A_PyA_Py, and investigated their duplex formations and the pyrene associations.

Synthesis and properties of new fluorescent nucleosides and oligodeoxynucleotides derived from 5-formyl-2'-deoxyuridine

Hirose, W., Sato, K., Matsuda, A. — 2009-09-12

5-Formyl-2'-deoxyuridine (fdUrd) is a product of the oxidation of thymidine and is known to induce mutation (A:T to G:C) in DNA. Therefore, a selective detection method for fdUrd is needed, but convenient methods have not been developed. We planned to develop a novel selective method to detect fdUrd in damaged DNA based on a specific fluorogenic derivatization of fdUrd using 2-aminothiophenol derivatives (ATs) as fluorogenic reagents. To achieve this goal, we first investigated the synthesis and fluorescence properties of some 5-(benzothiazol-2-yl)-2'-deoxyuridine derivatives (btdUrds). We also report the reaction between oligodeoxynucleotides (ODNs) containing fdUrd and ATs.

Fluorescent properties of acridonyl group in DNA duplex

2009-09-12

We designed and synthesized a nucleoside derivative in which the nucleobase is replaced with acridone. The nucleoside derivative was incorporated into an oligodeoxyribonucleotide (ODN), and its influence on the stability of ODN hybrids and its fluorescent properties in a DNA duplex were measured by thermodynamic analysis and fluorescent spectroscopy. These results showed that the acridonyl group could distinguish the type of nucleobase paired from fluorescent intensity, although the hybrid stability did not depend significantly on the types of nucleobases paired.

Optimization of the sequence of twisted intercalating nucleic acids (TINA) forming triple helix with the polypurine tract of the proviral HIV DNA

Boutorine, A. S., Doluca, O., Filichev, V. V. — 2009-09-12

Twisted intercalating nucleic acids form stable triplexes with polypurine tracts of double-stranded DNA. Their affinity depends on their length, primary structure and base contents, parallel or antiparallel orientation of oligonucleotides respectively to DNA, number of TINA residues and their relative positions. Basing on parallel CT, GT and antiparallel GT triplex-forming 16-mer oligonucleotides targeted to polypurine tract of HIV proviral DNA, we synthesized eleven different oligonucleotides with 2-4 TINA insertions in different positions. Studies of their interaction with target duplex by gel shift, fluorescence spectroscopy, circular dichroism and thermal denaturation demonstrated that antiparallel GT oligonucleotides form more stable triplexes than parallel TC or TG ones. Two best candidates were selected for the further studies. The first one (5'-AGGGxGGGTTTxTGTTTT-3', Kd = 219 nM) contains only two TINA insertions and does not aggregate in non-denaturing conditions, in contrast to majority of other oligonucleotides.

Design of extremely facile 3'- and 5'- ends free molecular beacons using C8 alkylamino substituted 2'-deoxyguanosine

2009-09-12

A novel fluorescent oligonucleotide probe containing pyrene-labeled C8 alkylamino-substituted 2'-deoxyguanosine and a practically useful 3'- and 5'- ends-free self-quenched molecular beacons (MB) were designed. A unique MB detectable by pyrene excimer fluorescence was also demonstrated.

Multipyrene tandem probes for detection of C677T polymorphism in MTHFR gene

2009-09-12

We designed tandems of oligo(2'-O-methylribonucleotides) conjugates containing two bispyrene (5'-bisPyr and 3'-bisPyr) groups on their junction for detection of C677T polymorphism in the methylenetetrahydrofolate reductase gene (MHTFR). The potential of SNP detection with multipyrene tandems of oligo(2'-O-methylribonucleotides) was demonstrated.

Synthesis and hybridization of oligonucleotides attached to a redox reporter via ethenyl linker at 5-position of pyrimidine base

Hasegawa, Y., Nakamura, M., Takada, T., Yamana, K. — 2009-09-12

We describe a new ferrocene-based redox-reporter that electronically couples to the DNA -electron system.

Immobilization of a naphthalene diimide-DNA complex on the gold through dithiolane moieties

Sato, S., Ohtsuka, K., Takenaka, S. — 2009-09-12

Naphthalene diimide 1 having two dithiolane moieties at its substituted termini was newly synthesized to immobilize double stranded DNA through dithiolane moieties of 1. Double stranded DNA could be immobilized on the gold surface of a quartz crystal microbalance (QCM) chip after binding 1. Once immobilized, the complex will serve as a hybridization marker based on its frequency decrease. QCM experiments showed that a DNA duplex with a 24-meric single stranded region also could be immobilized on the gold surface of a QCM chip and subsequent frequency decrease was observed after hybridization with a 24-meric complementary DNA, but not with non-complementary DNA, indicating specific hybridization. This result reveals that 1 provides a new immobilization method for intact DNA on gold and that the immobilized DNA can hybridize with target DNA.

Strategies for preparation of oligonucleotide biochips and their applications

Sethi, D., Kumar, P., Gupta, K.C. — 2009-09-12

In the last few years, DNA-microarray technology has emerged as a powerful tool for gene discovery, detection of mutations, and mapping. Here, we have developed a simple and efficient single-step method for immobilization of phosphoryl-, mercaptoalkyl-, thiophosphoryl- and aminooxyalkylated oligonucleotides onto an epoxylated glass surface. It resulted in higher immobilization and hybridization efficiency in comparison to those obtained with the standard method. The probes immobilized were found to be stable over a range of temperature and pH, suggesting that the chemistry could be used in integrated PCR/microarray devices. Also the immobilized spots were found to be of superior quality in terms of spot morphology, homogeneity and signal reproducibility. The constructed microarrays were successfully used for the discrimination of nucleotide mismatches and detection of bacterial diseases.

Evaluation of mobility of 2-N-tert-butylaminoxyladenosine incorporated into oligodeoxynucleotides

Higuchi, Y., Aso, M., Harada, R., Koga, N., Suemune, H. — 2009-09-12

We synthesized oligodeoxynucleotide (ODN, 3) containing 2-N-tert-butylaminoxyladenosine (1) and studied EPR spectra of 3 and its duplexes. The h₊/h₀ values in the EPR spectra of duplexes between 3 and 5-8 well correlated with Tm values. We also synthesized ODN 4 containing 2, which has a cyclic aminoxyl via a linker, to compare with ODN 3. The h₊/h₀ values in the EPR spectra of duplexes between 4 and 5-8 did not correlate with Tm values. These results indicate that 1 has a potential to monitor of motion of the nucleobase.

Real-time monitoring of mRNAs with fluorescence-modified RNA probes in living cells

Waki, R., Ueda, T., Yamayoshi, A., Kobori, A., Murakami, A. — 2009-09-12

In our previous study, we reported that bispyrene-modified RNA probes (OMUpy2) were useful for RNA detection in homogeneous physiological media^1-2. The aim of this study is to establish in situ monitoring system to detect mRNAs in living cells by OMUpy2. We chose c-fos mRNA as a target RNA which is known as one of the immediate-early genes. The real-time moni- toring of mRNAs was carried out in living C4II cells. After transfection of OMUpy2 into the cells which had been incubated with serum-free medium, cells were stimulated by media with serum and the resulting expression of mRNA was monitored by a fluorescence microscope. In the case of OMUpy2-CF3S, which was complementary to c-fos mRNA, fluorescence emission around 480 nm was observed obviously.

These results suggest that mRNA expressed by the serum stimulation was successfully visualized in a real-time manner.

Synthesis of exciton-controlled fluorescent probes for RNA imaging

Ikeda, S., Kubota, T., Yanagisawa, H., Yuki, M., Okamoto, A. — 2009-09-12

The excitonic interaction of thiazole orange dyes is known to suppress fluorescence emission. We have developed hybridization-sensitive fluorescent probes utilizing the excitonic interaction of two thiazole orange dyes connected to the probes. Here, we report nuclease-resistant hybridization-sensitive probes for long-term intracellular RNA imaging.

Protein detection using oligonucleotide probes

Shibata, A., Abe, H., Furukawa, K., Tsuneda, S., Ito, Y. — 2009-09-12

We developed a new nucleic acid-based fluorescence probe for protein detection. The method is based on the scission of an aptamer into two probes, which are then attached with a chemically reactive fluorogenic compound. The protein-dependent association of the two probes accelerates a chemical reaction and indicates the presence of the target protein, which is detected using a fluorescence readout. The arginine-rich motif peptide was targeted by this type of probe. In presence of the peptide, the fluorescence signal at 450 nm increased, and no significant increase in fluorescence was observed in the absence of the peptide. An oligonucleotide-based fluorescence probe was successfully applied to the detection of the ARM peptide in solution.

Promotion of triplex formation by 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification

Sasaki, K., Rahman, S. M. A., Sato, N., Obika, S., Imanishi, T., Torigoe, H. — 2009-09-12

We examined the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid (3'-amino-2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 3'-amino-2',4'-BNA modified TFO was significantly higher than that observed with unmodified TFO. The 3'-amino-2',4'-BNA modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 3'-amino-2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Promotion of strand invasion by utilizing entropically-favored PNA

Ishizuka, T., Sforza, S., Komiyama, M. — 2009-09-12

Invasion of peptide nucleic acid (PNA) is important to target a specific site in double-stranded DNA. However, it needs improvement in many ways such as sequence versatility and invasion efficiency. It is here reported that one 10-mer strand of -lysine substituted peptide nucleic acid (PNA) efficiently invades double-stranded DNA (dsDNA) in the presence of single-strand binding protein (SSB) via Watson-Crick rule. According to the thermodynamic parameters in this study, highly efficient invasion was achieved by a favorable entropy change. The importance of preorganization of PNAs for efficient strand invasion is indicated.

Development of new DNA triplex triads by using 5-substituted deoxycytidine

Kanamori, T., Tsunoda, H., Ohkubo, A., Sekine, M., Seio, K. — 2009-09-12

We developed a new artificial DNA triplex triad which has the adenine nucleobase in the first strand. This triplex triad (A--C*) is composed of adenine, pseudouridine and 5-sudstituted cytosine as the 1st, 2nd and 3rd strand nucleobases, respectively. The molecular design and synthesis of the nucleobases for the 2nd and 3rd strand of the triplex are also described.

Synthesis and thermal denaturation studies of covalently linked DNA triplexes

Okamoto, I., Ito, S., Ono, T., Ono, A. — 2009-09-12

We report on the synthesis and thermal stability of small covalently linked DNA triplexes. These modified triplexes were found to contain covalently linked T–T pairs at the edges, and thermal denaturation studies revealed that the covalent linking efficiently stabilized triplex formation.

Evaluation of the antisense effect of PEGylated oligodeoxynucleotides containing intelligent nucleoside analogues

Taniguchi, Y., Kurose, Y., Nishioka, T., Nagatsugi, F., Sasaki, S. — 2009-09-12

We have previously reported that the 2-amino-6-vinylpurine (AVP) in oligonucleotide (ODN) showed the highly selective and effective cross-linking reaction toward cytosine within target complementary sequences. Recently, we revealed that the PEGylated ODN containing AVP analogues exhibited the effective antisense effect in the cultured cell. However, subsequent studies showed that the cross-linking ability of AVP for cytosine was affected by the target RNA sequences. We therefore developed new intelligent nucleoside analogues connected AVP to sugar through the ethylene linker (et-AVP), that exhibited highly effective cross-linking ability to rC in the target RNA. In this paper, we described the synthesis of the PEGylated ODN containing cross-linking nucleoside analogues and properties of it about RNase H activity and antisense effect in cell lysate. As a result, the PEGylated ODN containing et-AVP showed the effective antisense effect in the non-cell system for targeting the luciferase mRNA by non-RNase-H mechanism.

Design and synthesis of the novel cross-linking agent

Kusano, S., Hattori, K., Imoto, S., Nagatsugi, F. — 2009-09-12

Previously, we have developed a highly efficient and selective cross-linking reaction to the cytosine base at the target site of DNA using the oligodeoxynucleotide (ODN) containing 2-amino-6-vinylpurine derivative (1). Based on these results, we have designed the novel cross-linking agents, which are pyrimidine derivatives having two hydrogen bond sites and vinyl group as a reactive moiety. In this paper, we wish to report the results to investigate on the synthesis of the pyrimidine derivatives having potential as novel cross-linking agents.

DNA cross-link generated by a novel modified DNA containing a formyl group

Shibata, T., Dohno, C., Nakatani, K. — 2009-09-12

We here report the synthesis of novel modified nucleic acid containing a formyl group and the evaluation of interstrand cross-linking between the modified DNA and the complementary strand. The synthesis of the formyl-containing oligodeoxynucleotide (ODN) was achieved by a post synthetic modification of corresponding ODN containing a 1,2-diol. The ODN containing the diol moiety was successfully synthesized by a standard phosphoramidite method, and was quantitatively converted to the desired formyl-containing ODN by sodium periodate oxidation. Hybridizaiton of the formyl ODN and the complementary ODN produced the interstrand cross-link between the formyl group and N⁶-exocyclic amino group of adenine.

DNA ligation using photoremovable protecting groups

Kawano, Y., Takada, T., Nakamura, M., Yamana, K. — 2009-09-12

Template-directed ligation of oligonucleotides by photochemical reaction has attracted much interest because of its biomedical and synthetic applications. In this study, we developed photoligaton of DNA by using a photoremovable protecting group and thiol-disulfide exchange reaction. A phosphoroamidite of o-nitrobenzyl derivatives were synthesized, and DNA modified with a nitrobenzyl-protected thiol group and disulfide group was synthesized by conventional phosphoroamidite chemistry using a DNA synthesizer. It was shown that photochemical reaction of a nitrobenzyl group with UV irradiation produced a free thiol group, leading to the DNA ligation through the thiol-disulfide exchange reaction.

Handy and prompt DNA separation using PNA with internal disulfide bond

Aiba, Y., Komiyama, M. — 2009-09-12

PNA (peptide nucleic acid) is a DNA analogue which has a peptide backbone. PNA is very useful as a recognition probe because of its immensely high affinity to DNA. In this study, we used PNA as a DNA separation tool. Furthermore, we introduced a cleavable disulfide bond into main chain of PNA in order to facilitate the removal of PNA from PNA/DNA duplex. This disulfide bond was readily cleaved by various reducing agents and then the resultant two short PNA fragments could not form stable PNA/DNA duplex anymore. Accordingly, a desired DNA fragment was picked up from the DNA mixtures and readily recovered by the reductive treatment.

Radiolytic one-electron reductive cyclization of oligodeoxynucleotides possessing a disulfide bond

Matsumoto, E., Tanabe, K., Nishimoto, S.-i. — 2009-09-12

Radiolytic one-electron reduction of oligodeoxynucleotides (ODNs) possessing a disulfide bond induced strand exchange reaction in a hypoxia selective manner. In this study, we attempted to apply this radiolytic reaction to stem-and-loop structured ODNs. Hypoxic X-irradiation to an aqueous solution of stem-and-loop structured ODNs produced a cyclized DNA in good yield via an intrastrand ligation reaction.

Development of a novel method to determine the concentration of heavy metal cations: Application of the specific interaction between heavy metal cation and mismatch DNA base pair

Kozasa, T., Miyakawa, Y., Fukushi, M., Ono, A., Torigoe, H. — 2009-09-12

We have already found that Hg(II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4 °C. We have also found that Ag(I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4 °C. Using the specific interaction, we developed a novel sensor to determine the concentration of each of Hg(II) and Ag(I) cation. The sensor is composed of a dye-labelled T-rich or C-rich DNA oligonucleotide, F2T6W2D: 5'-Fam-T₂CT₂CT₂C₄T₂GT₂GT₂-Dabcyl-3' or F2C6W2D: 5'-Fam-C₂TC₂TC₂T₄C₂AC₂AC₂-Dabcyl-3', where 6-carboxyfluorescein (Fam) is a fluorophore and Dabcyl is a quencher. The addition of Hg(II) cation decreased the intensity of Fam emission of F2T6W2D at 520 nm in a concentration-dependent manner. Also, the addition of Ag(I) cation decreased the intensity of Fam emission of F2C6W2D at 520 nm in a concentration-dependent manner. We conclude that, using the novel sensor developed in this study, the concentration of each of Hg(II) and Ag(I) cation can be determined from the intensity of Fam emission at 520 nm.

Electronic structure and UV absorption spectra of metal-mediate DNA: an approach from theoretical chemistry

2009-09-12

We theoretically evaluated the stability, UV-Vis spectra and possibility of stacking of [S-M(II)-S] (M=Ni, Pd, Pt, S: hydroxypyridonethione) by means of the density functional theory (DFT). From the view of the free energy, we assessed formation energy of possible combinations of chalcogen atoms and metal cations. The results confirmed that [H-Ni(II)-H] and [S-Cu(II)-S] would form stable metal-base pairing, on the other hand [H-Pt(II)-H] would not, which have been experimentally proven. Moreover, by use of time-dependent density functional theory (TDDFT), we observed d-* transition accompanied with -* transition in [S-M(II)-S]. These results reveal that metal-to-ligand charge transfer (MLCT) shifts the peak of -* transition in [S-2H⁺-S] (without metal cations).

Acceleration of guanine oxidation under visible light irradiation by photon upconversion based on triplet-triplet annihilation

Tanaka, K., Kitamura, N., Inafuku, K., Chujo, Y. — 2009-09-12

We report the fluorescent polymer complex which can show fluorescence emission at 380 nm with the excitation of 520 nm in aqueous media. This photon upconversion based on triplet–triplet annihilation can efficiently take place via inter-molecular energy transfers between the Ru complex as a sensitizer and anthracene molecules as an emitter captured into the water-soluble network polymers. We performed the oxidation reaction of 2'-deoxyguanosine by riboflavin in the presence of the polymer complex with the visible light irradiation. It was clearly indicated that oxidative decomposition can be accelerated by UV light generation via upconversion based on triplet–triplet annihilation.

Photochemical generation and reaction of 2'-substituted analogues of C4'-oxidized abasic lesion

2009-09-12

The C4'-oxidized abasic site (1) is one of the oxidatively damaged DNA lesions induced by antitumor bleomycins. The reactivity of 1 with amine under neutral conditions giving lactam 2 and its structural similarity to unmodified DNA suggested the possibility that ODN containing 1 could react with proteins which interact with DNA at lysine residue. In order to provide nucleic acid analogues with useful lysine modifying reactivity, we planned to study reaction of 2'-substituted analogues of 1, such as 3-5, with amine and their corresponding caged precursors 6-8 were synthesized. Uncaging reaction of 16 containing 6 proceeded efficiently. The reaction of obtained 17 containing 3 with Z-Lys-OH did not afford products corresponding to lactam. However, HPLC analysis of the reaction mixture suggested interaction of 3 with Z-Lys-OH.

Development of light-responsive BNA toward the spatiotemporal control of gene expression

Morihiro, K., Kodama, T., Nishida, M., Imanishi, T., Obika, S. — 2009-09-12

We designed and synthesized light-responsive BNA (bridged nucleic acid), which has a photolabile group at the bridged structure. Light-Responsive BNA can change its structure by a two stages process: photoirradiation followed by nucleophilic treatment. In oligonucleotides containing light-responsive BNA moieties, binding affinity with complementary single-stranded RNA was lost upon photoirradiation, but could be restored by nucleophilic treatment.¹

Line up base pairs and intercalators one by one in a stable duplex

Liang, X., Mochizuki, T., Nishioka, H., Asanuma, H. — 2009-09-12

A stable double helix involving alternating base pairs and azobenzene moieties was constructed. In this supramolecule, base pairs and azobenzenes are lined up one by one to form an interstrand-wedged motif: each base pair is sandwiched with two azobenzenes, and each azobenzene intercalates between two base pairs. This motif was formed by the hybridization of two modified DNA tethering multiple azobenzene moieties at a frequency of one azobenzene for every two nucleotides. Furthermore, this structure could be simply dismantled by UV light irradiation and reformed with the irradiation of visible light. By using this new duplex motif, construction of a variety of photoresponsive nanostructures and nanodevices is highly expected.

Regulation of duplex DNA strand displacement by visible light sensitive bis-peptide nucleic acid

Sawada, S., Imada, I., Kato, N., Kaihatsu, K. — 2009-09-12

A novel visible light sensitive azobenzene (AZO) was synthesized and introduced into bis-peptide nucleic acid (bis-PNA), which consists of two homopyrimidine PNA strands, as a linker. Visible light irradiation of the bis-PNA-AZO conjugate induced photoisomerization of the azobenzene moiety from trans to cis. The cis-form of bis-PNA-AZO displaced the complementary duplex DNA more efficiently than the trans-form.

Photo-switching of vinylpyrene-substituted 2'-deoxyguanosine and its application

Saito, Y., Takeuchi, Y., Matsumoto, K., Takahashi, N., Suzuki, A., Saito, I. — 2009-09-12

We synthesized C8-vinylpyrene-substituted 2'-deoxyguanosine ^VPyG and studied the photoinduced reversible E–Z isomerization. When E-isomer was irradiated with visible light (>420 nm), E- to Z-isomerization took place very rapidly, while upon irradiation with UV-light (~365 nm), Z-isomer was converted to E-isomer. When Z-isomer was illuminated with 365– 400 nm light, no fluorescence was observed, while E-isomer showed a very strong fluorescence emission, indicating that ^VPyG could be a useful fluorescence switching molecule.

Photo-controllable aptamer

Ogasawara, S., Maeda, M. — 2009-09-12

We have successfully developed a new method for photoregulation of G-quadruplex formation using cis-trans photoisomerization of the photochromic nucleobase ^8FVG. Our photo-controllable quadruplexes can be switched between a very stable quadruplex state and a non-structured state in a straightforward and reversible fashion. We also demonstrated reversibly control binding of a G-quadruplex aptamer to thrombin.

Development of a rapid and reversible photocrosslinking of RNA

Yoshimura, Y., Ohtake, T., Okada, H., Fujimoto, K. — 2009-09-12

We describe a novel reversible interstrand photocrosslinking reaction of RNA. In this system, a modified oligodeoxynucleotide (ODN) containing 3-cyanovinylcarbazole nucleoside (^CNVK) reacts by photoirradiation at 366 nm for 1 s with pyrimidine residue of a complementary template RNA to yield a crosslinked product in 94% yield.

A selective and sensitive detection of SNP between rice cultivars by using DNA photoligation

Yoshimura, Y., Okada, H., Ohtake, T., Tsukaguchi, T., Fujimoto, K. — 2009-09-12

We describe a selective and sensitive single-nucleotide polymorphism (SNP) typing of genes from rice cultivars by using DNA photoligation. This SNP typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity.

Photoinduced electron transfer reaction in diaminostilbene-tethered DNA duplexes

Ito, T., Hayashi, A., Uchida, T., Tanabe, K., Yamada, H., Nishimoto, S.-i. — 2009-09-12

DNA duplexes containing diaminostilbene (DAS) as a photoinduced electron donor were synthesized to investigate mechanisms of electron injection into DNA and the succeeding electron transfer in the duplexes. DAS-Capped hairpin DNA showed a high structural stability thereby attains large interaction between DAS and the terminal base pair. DAS-Tethered DNA by a single linker at the end of the duplex was also synthesized and the yields of photoinduced electron transfer through mismatched base pairs were quantified. Both duplexes showed similar electron transfer efficiencies depending on the base pairs, which suggests DAS stacks well on the "-way" of the duplex DNA.

A photochemical detection of methylcytosine by using hydrophobic interaction

Ami, T., Ogino, M., Taya, Y., Takemura, Y., Fujimoto, K. — 2009-09-12

In this paper, the nonenzymatic detection of 5-methylcytosine with high selectivity is described. We present a new methylation detection method by using template-directed photoligation through 5-cyanovinyl-2'-deoxyuridine (^CU). Significantly, the photoligation yield of the 5-methylcytosine case was approximately 9.1-fold higher than that in the case of cytosine.

Photoelectrochemical identification of 5-methylcytosine modification in DNA: combination of photosensitization and enzymatic cleavage

Tanabe, K., Yamada, H., Ito, T., Nishimoto, S.-i. — 2009-09-12

We present a novel photoelectrochemical approach for discriminating between cytosine and 5-methylcytosine in DNA when used in combination with enzymatic digestion. A photosensitizer-linked DNA duplex bearing 5-methylcytosine or cytosine at a given restriction site of the strand was immobilized on a gold electrode and digested with enzyme, and the photocurrent response was measured. We observed high photocurrent density that was selective for the methylated duplex.

Osmium complex binding to mismatched methylcytosine: effect of adjacent bases

Nomura, A., Tainaka, K., Okamoto, A. — 2009-09-12

We investigated the efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes. Osmium complexation was not observed in fully matched duplexes, whereas the complexation site and efficiency in mismatched duplexes depended on the 5'-neighboring base of the 5-methylcytosine. In particular, when the base adjacent to the 5' side of the mismatched base pair was thymine, a unique side reaction was observed. However, the mismatched base pairs did not influence the selectivity of osmium complexation with methylated DNA.

Efficient photooxidative strand cleavage at 5-methylcytosine in DNA by sensitization with 9,10-anthraquinone-tethered oligonucleotides

Yamada, H., Kitauchi, Y., Tanabe, K., Ito, T., Nishimoto, S.-i. — 2009-09-12

Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of 5-methylcytosine (^mC)-containing DNA and 9,10-anthraquinone (AQ)-tethered complementary oligodeoxynucleotide led to selective oxidative strand cleavage at the target ^mC site, whereas no strand cleavage was observed for the duplex containing normal cytosine instead of ^mC. Incorporation of an AQ sensitizer into the interior of a strand induced an enhanced one-electron photooxidation, presumably because of a much larger intersystem crossing efficiency, leading to an efficient strand cleavage at the target ^mC site in DNA. Optimization of the photosensitizer could afford more strand cleavage at ^mC in DNA, thereby allowing for the more sensitive detection of the target ^mC on a sequencing gel.

Analysis of an RNA adduct formed from aminophenylnorharman

2009-09-12

The endogenous mutagenic/carcinogenic 9- (4'-aminophenyl) -9H- pyrido [3,4-b] indole (aminophenylnorharman, APNH) is formed from norharman and aniline in the presence of cytochrome P-450s. The major APNH-DNA adduct has been reported to be 2'-deoxyguanosin-8-yl-aminophenylnorhaman (dG-C8-APNH). In addition, demonstrated formation of APNH-RNA adduct and conducted a structural analysis using various spectrometric approaches. The compound produced from guanosine (Guo) and N-acetoxy-APNH, an ultimate mutagenic form of APNH, was concluded to be guanosin-8-yl-APNH (Guo-C8-APNH) on the basis of various spectroscopic analysis. The same adduct was found in the livers of rats administered APNH. The total adduct levels of APNH-RNA were six times higher than total APNH-DNA adducts in the same rat liver samples.

Fluorescence detection of the endonuclease III reaction using modified oligonucleotides

2009-09-12

We have developed a method for the detection of the endonuclease III reaction by fluorescence. The probes were 13-base-pair hairpin-shaped oligonucleotides containing one of the isomers of thymine glycol or 5,6-dihydrothymine as a damaged base at the center, and had a fluorophore and a quencher at the 5' and 3' ends, respectively. Fluorescence was detected when the probe was cleaved by the enzyme, because the short fragment bearing the fluorophore could not be hybridized to the quencher strand at the incubation temperature. The substrate specificity was shown using Escherichia coli and human enzymes.

Reaction of cytosine with bisulfite and hydroxylamine

Oka, Y., Takei, F., Nakatani, K. — 2009-09-12

The synthesis of DNA oligomer containing N⁴-hydroxy-5,6-dihydrocytosine-6-sulfonate by the simultaneous addition of bisulfite and hydroxylamine under mild conditions is reported. To the best of our knowledge, the reaction of the oxime into the ketone, N⁴-hydroxy-5,6-dihydrocytosine-6-sulfonate into 5,6-dihydrouracil-6-sulfonate or uracil has not been reported. Moreover, this issue contains the difficulty in specific reactivity of oxime derivative from DNA bases include ^5mC toward reagent; for example, T also reacts to potassium permanganate known as a reagent for oxime into ketone. Here we examined the transformation of DNA containing N⁴-hydroxy-5,6-dihydrocytosine-6-sulfonate into 5,6-dihydrouracil 6-sulfonate and the following elimination of sulfonate.

Progress in the bisulfite modification of nucleic acids

Hayatsu, H., Negishi, K., Wataya, Y. — 2009-09-12

Bisulfite modification is a principal tool for analyzing DNA methylation, the methyl substitution at position 5 of cytosine residues. Hypermethylation is known to cause silencing of genes, which may result in cell function failures. DNA methylation analysis is therefore a focus of attention in various fields of biological sciences, including even clinical practices for treatment of cancer patients. In 2004, we reported that the bisulfite modification of DNA necessary in this analysis can be speeded up significantly by using a high concentration ammonium bisulfite solution (10 M), in place of traditional sodium bisulfite solution of 5 M concentration. Evaluations on this newer protocol have now come out from several laboratories, showing that this quick process can yield results with greater accuracy compared to those obtainable with widely-practiced low-concentration methods. Another aspect reported here is a study on the desulfonation of uracil-bisulfite adduct to form uracil, the last step of the bisulfite-conversion of cytosine to uracil. Kinetic measurements for the desulfonation of uridine-bisulfite adduct at a near-neutral pH region are described.

The oxidation of 2'-deoxy-8-oxoguanosine by iodine

2009-09-12

8-Oxoguanine is the typical oxidative product, but 8-oxoguanine is further oxidized by several oxidizing agents. Here, we investigated the oxidation of 2'-deoxy-8-oxoguanosine with aqueous iodine.

Recognition and reaction mechanisms of the (6-4) photolyase as determined by using a (6-4) photoproduct analog

Yamamoto, J., Hitomi, K., Hayashi, R., Getzoff, E. D., Iwai, S. — 2009-09-12

The (6–4) photoproduct, which is one of the major UV-induced DNA lesions formed at bipyrimidine sites, causes carcinogenesis at high frequency. The (6–4) photolyases restore the (6–4) photoproducts to their intact bases in a light-dependent manner, but its overall repair mechanism remains obscure. To investigate the light-dependent conversion of the (6–4) photoproduct, we prepared a (6–4) photoproduct analog with modification at 3' pyrimidone ring, in which the carbonyl group was replaced with an imine to apply to the (6–4) photolyase assay. The (6–4) photolyase shows affinity to an oligonucleotide carrying this imine analog of the (6–4) photoproduct, though the imine analog is not repaired by the (6–4) photolyase.

Roles of specialized DNA polymerases in mutagenesis by oxidized guanine

Kamiya, H., Satou, K., Hori, M., Yamaguchi, A., Harashima, H. — 2009-09-12

To assess the involvement of specialized DNA polymerases in the mutagenesis induced by an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), shuttle plasmid DNA containing the supF gene and 8-OH-dGTP were introduced into human 293T cells in which specialized DNA polymerases were knocked-down by siRNAs. The knock-downs of DNA polymerases and , and REV1 reduced the A:T->C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A. In contrast, the knock-down of DNA polymerase did not affect the mutations induced by 8-OH-dGTP. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols plus and REV1 plus DNA pol (but not by that of DNA pol plus REV1), suggesting that REV1-DNA pol and DNA pol work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by 8-OH-dGTP.

Comparison of the activities of bacterial and mammalian nucleotide excision repair systems for DNA-protein crosslinks

Nakano, T., Salem, A. M.H., Terato, H., Pack, S. P., Makino, K., Ide, H. — 2009-09-12

Endogenous and environmental genotoxic agents produce DNA damage and induce cell death and mutations. The repair mechanisms of base lesions and single and double strand breaks have been well characterized in both prokaryotic and eukaryotic cells. However, the molecular pathways that repair or tolerate DNA-protein crosslinks (DPCs) remains to be largely elucidated. In this study, we constructed DNA substrates containing defined DPCs and assessed the incision activities of prokaryotic and eukaryotic nucleotide excision repair systems for DPCs in vitro.

Accurate curve fitting procedure for UV melting analysis of highly thermostable RNA hairpins

Kawakami, J., Tanaka, Y., Kishimoto, K. — 2009-09-12

Thermostable RNA hairpins with specific loop sequences are critically important structural elements for proper folding of functional RNAs. Thermodynamic parameters of the structural elements are indispensable for secondary structure prediction. However when RNA folds into highly thermostable hairpin-loop structures, it is difficult to determine the melting temperature and other thermodynamic parameters from the normal UV melting analysis. In this study, we demonstrate a procedure to solve the problem using our software, Jfit. UV absorbance in the higher temperature region above 95°C and the upper baseline were estimated using this analytical procedure. The ideal lines of –RlnK vs. 1/T were generated from the predicted thermodynamic parameters and the solution candidates were compared with the van’t Hoff plot generated from the measured melting curve to judge their self-consistency. As a result, our analysis was able to reduce the number of the possible solution and to specify the best set of thermodynamic parameters for the stable hairpin formation.

Investigations of the cation binding to nucleotides by monitoring the hairpin-duplex equilibrium of a self-complementary sequence

Nakano, S.-i., Hirayama, H., Sugimoto, N. — 2009-09-12

Nucleotide folding accompanies cation binding that shields the electronegative potential of phosphate groups, and metal ions in the condensation layer predominantly associate diffusely with base-paired nucleotides. Although metal ions bound at specific sites have been well studied, information of diffusely bound cations, that usually have a weak binding affinity than those associating at specific sites, have not been thoroughly studied. We explored a convenient experimental system using a self-complementary nucleotide sequence for analyzing cationic ligands diffusely bound to DNA or RNA base pairs. To study the metal ion-nucleotide interaction under a non-homologous aqueous condition, solutions containing a large amount of PEG (polyethylene glycol) were examined. We found that PEG (e.g., 20 wt%) substantially influenced the metal ion binding to nucleotides, suggesting significances of the molecular environment on nucleotide-cation interactions.

Synthesis of potent G-quadruplex binders of macrocyclic heptaoxazole and evaluation of their activities

Tera, M., Iida, K., Shin-ya, K., Nagasawa, K. — 2009-09-12

Guanine-rich DNA sequences form unique three-dimensional conformation known as G-quadruplexes (G-q). G-q structures have been found in telomere and in some oncogene promoter. Recently, it was suggested that G-q showed some biological activities including telomere shortening and transcriptional regulation. In this paper, we synthesized selective G-q binders and evaluated of their biological activities.

G-quadruplex recognition by macrocyclic hexaoxazole (6OTD) dimer

Iida, K., Tera, M., Shin-ya, K., Nagasawa, K. — 2009-09-12

Telomestatin (TMS: 1) is as a potent and selective telomeric G-quadruplex binder. Two molecules of TMS were suggested to be intercalated to one telomeric G-quadruplex according to docking study. In this paper, we designed and synthesized hexaoxazole TMS derivative (6OTD) dimer, and evaluated its G-quadruplex stabilizing ability.

Tetraplex structure of budding yeast telomeric DNA

Torigoe, H., Horio, E., Takehara, T., Kaneda, K., Kozasa, T. — 2009-09-12

We examined the structural properties of budding yeast telomeric DNA sequences, SCTELG4: 5'-(TGGG TGTG)₄-3' and SCTELGG4: 5'-(TGGGTGTGG)₄-3', in the presence of Na⁺ or K⁺. The conformation of SCTELG4 with Na⁺ was a mixture of parallel tetraplex DNA and unstructured single-stranded DNA. On the other hand, the conformation of SCTELGG4 with Na⁺ was a mixture of parallel and antiparallel tetraplex DNA. The ratio of the amount of parallel tetraplex to that of unstructured single-strand was increased for SCTELG4 by the presence of K⁺. The conformation of almost all of SCTELGG4 was changed into parallel tetraplex by the presence of K⁺. These results indicate that structural change of SCTELG4 and SCTELGG4 may be induced by the type of cation. We conclude that the conformation of budding yeast telomeric DNA sequences depends on the base sequence and the type of cation.

Thermal stability and hydration state of DNA G-quadruplex regulated by loop regions

Fujimoto, T., Miyoshi, D., Tateishi-Karimata, H., Sugimoto, N. — 2009-09-12

We designed and synthesized DNA oligonucleotides that were able to fold into intramolecular DNA G-quadruplexes with different number of G-quartet planes and thymines in the each loop region [GmTn: m indicates the number of G-quartet planes (2 ≤ m ≤ 4), and n indicates the number of thymines in the each loop region (2 ≤ n ≤ 4)]. Effects of molecular crowding on structure of G-quadruplexes were investigated by circular dichroism spectra in buffers containing 0 wt% or 40 wt% PEG200 (poly(ethylene glycol) with an average molecular weight of 200). The results showed that G2T2, G2T3, G2T4 and G3T4 folded into antiparallel G-quadruplexes under the dilute and molecular crowding conditions. On the other hand, G3T2, G3T3, G4T2, G4T3, and G4T4 underwent structural transitions from antiparallel or mixture to parallel G-quadruplex by the addition of PEG200. These result and thermodynamic analysis of G2Tn demonstrated that loop regions had pivotal roles to determine structure and stability of G-qudruplexes.

Human telomeric RNA r(UAGGGU) sequence forms parallel tetraplex structure with U-quartet

Kimura, T., Xu, Y., Komiyama, M. — 2009-09-12

Recent studies showed that telomere DNAs are transcribed into telomeric-repeat-containing RNAs in mammalian cells and these RNAs are associated with the chromosome ends. Therefore, they may be key components of telomere machinery. We have reported that the human telomeric RNA sequence forms parallel G-quadruplex structure in the presence of Na⁺ ions. In the current study, using a combination of CD and NMR experiments, we found that the human telomeric RNA r(UAGGGU) sequence can form parallel tetraplex structure with U-quartet in the presence of K⁺ or Na⁺ ions. These results provide valuable information to allow understanding of the molecular basis of human telomeric RNA structure.

Characterization of heme coordination structure in heme-DNA complex possessing gaseous molecule as an exogenous ligand

Saito, K., Nakano, Y., Tai, H., Nagatomo, S., Hemmi, H., Mita, H., Yamamoto, Y. — 2009-09-12

We have previously demonstrated that heme, iron(III)-protoporphyrin IX complex, and a parallel-quadruplex DNA assembled from d(TTAGGG) form a stable coordination complex called "heme-DNA complex". The heme-DNA complex exhibits a variety of spectroscopic characteristics remarkably similar to those of met-form of myoglobin, oxygen-binding hemoprotein, reflecting that the heme environments in the two systems are highly alike to each other. In a course of our effort toward exploring functional properties of the heme-DNA complex, we have investigated binding of gaseous molecules such as CO and O₂ to the heme-DNA complex. The present study revealed that the heme-DNA complex exhibits ability to accommodate these molecules as exogenous ligands.

Interaction between tetraplex structure of yeast telomeric DNA and tetraplex-binding ligand

Torigoe, H., Horio, E., Takehara, T., Kaneda, K., Kozasa, T. — 2009-09-12

Fission yeast telomeric DNA sequence, SP4G4: 5'-(GGGGTTAC)₄-3' has been reported to form the antiparallel tetraplex in the presence of K⁺. We examined the structural properties of budding yeast telomeric DNA sequences, SCTELG4: 5'-(TGGGTGT G)₄-3' and SCTELGG4: 5'-(TGGGTGTGG)₄-3', in the presence of K⁺. The major conformation of SCTELG4 and SCTELGG4 was a parallel tetraplex DNA. We also examined the interaction between tetraplex-binding ligand, TMPyP4, and each of the antiparallel tetraplex of SP4G4 and the parallel tetraplex of SCTELG4 and SCTELGG4. The ability of TMPyP4 to bind with all of the tetraplexes was observed. We conclude that TMPyP4 has the ability to bind with both the parallel and antiparallel tetraplex of the telomeric DNA sequences from budding and fission yeasts.

Interaction between tetraplex structure of mouse telomeric DNA and telomeric DNA binding domains of mouse telomere binding protein Pot1

Kaneda, K., Torigoe, H. — 2009-09-12

Mouse telomeric DNA sequence, Tel3.5: 5'-AGGG(T TAGGG)₃-3', has the ability to form antiparallel tetraplex structure in the presence of Na⁺. We examined the interaction between the antiparallel tetraplex structure of Tel3.5 and each of two single-stranded telomeric DNA-binding domains of mouse telomere binding protein Pot1, mPot1OB1 and mPot1OB2. The antiparallel tetraplex of Tel3.5 became unfolded upon the interaction with mPot1OB1. On the other hand, no significant structural change of the antiparallel tetraplex of Tel3.5 was observed upon the interaction with mPot1OB2. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of mPot1OB1 to unfold the antiparallel tetraplex of the mouse telomeric DNA is required for telomerase-mediated telomere elongation.

Identification of DNA binding specificity for TLS

Takahama, K., Arai, S., Kurokawa, R., Oyoshi, T. — 2009-09-12

TLS (Translocated in liposarcoma) has been characterized as a rearranged gene in chromosomal translocations specific of human myxoid liposarcoma. The various cellular functions of TLS participating either in transcription or splicing processes are thought the involvement of an interaction of TLS with DNA and/or RNA. To investigate insight into DNA-TLS interaction, we performed Electrophoretic mobility shift assay of TLS with G-quadruplex DNA. It revealed that TLS especially bound to single stranded human telomeric DNA in the presence of potassium ion while it was not able to bind double stranded human telomeric DNA and single stranded human telomeric DNA in the presence of sodium ion.

Influence of 3'-azido-2',3'-dideoxyguanosine treatment on telomere length in human telomerase-immortalized human fibroblast cells

Kurashina, Y., Liu, X., Kato, C., Inoue, N., Saneyoshi, M., Yamaguchi, T. — 2009-09-12

In order to study telomerase activation in normal cells, a telomerase-immortalized fibroblast cell line, hTERT-BJ1, treated with a telomerase inhibitor, 3'-azido-2',3'-dideoxyguanosine (AZddG), is considered to be a good model. Long-term treatment with AZddG resulted in telomere shortening from 10-20 kbp to 5-6 kbp in cultured hTERT BJ1 cells. However, the telomere length then stabilized. As expected, removal of AZddG from the culture medium induced telomere lengthening in the cells, suggesting that telomerase activity was recovered upon AZddG removal. The effect of recovery of telomerase activity in hTERT-BJ1 cells will be investigated in future studies.

Design of cationic graft copolymers as a potential inducer of B-Z transition

Shimada, N., Kano, A., Maruyama, A. — 2009-09-12

Biological roles of transition from B form to Z form of DNA (B-Z transition) have recently received attention. The B-Z transition was also employed as driving machinery of a nano-mechanical DNA device. However, there are little reports of effective inducer of the B-Z transition. We previously reported that poly(_L-Lysine)-graft-dextran induces B-Z transition and grafted dextran plays an important role for the B-Z transition. In this report, we designed cationic graft copolymer as a potential inducer of B-Z transition. Series of the copolymers consisting of poly(_L-Lysine) backbone and abundant of dextran (Dex) or poly(ethylene glycol) (PEG) side chains were prepared. The B-Z transition of poly(dG-dC)/Poly(dG-dC) were observed in the presence of these copolymers. The copolymers having higher content of Dex or PEG effectively induced the B-Z transition compared to that having lower content. The result indicated that not only electrostatic interaction between DNA and the poly(_L-Lysine) backbone but also hydrophilic graft chains play a role for the B-Z transition. We speculated that Dex- or PEG-enriched environment is favorable for Z form.

Release of DNA binary complexes from the ternary complexes by carboxymethyl Poly(L-histidine)

Asayama, S., Sudo, M., Kawakami, H. — 2009-09-12

The DNA ternary complexes with carboxymethyl poly(l-histidine) (CM-PLH) and poly(ethylenimine) (PEI) have released the DNA binary complexes with PEI by the protonation of CM-PLH at endosomal/lysosomal pH. The dissociation of the CM-PLH from the CM-PLH/PEI/DNA ternary complexes is proved by the fluorescence resonance energy transfer (FRET) analysis between the CM-PLH and PEI. The resulting PEI/DNA binary complexes easily released DNA, as compared with the CM-PLH/PEI/DNA ternary complexes, which was examined by competitive exchange with dextran sulfate. The release of the DNA binary complexes from the ternary complexes is promising mechanism for higher transfection activity by the CM-PLH/PEI/DNA ternary complexes.

Acridone-labeled DNA aptamer for the detection of biomolecules

Ozaki, H., Hagiwara, Y., Asakura, H., Kuwahara, M. — 2009-09-12

An acridone-labeled DNA aptamer was synthesized to produce an aptamer probe. This aptamer probe could detect a specific molecule based on conformational changes upon binding of the specific molecules and the quenching of acridone emission.

Covalently linked fluorescent ribonucreopeptide sensors

Fukuda, M., Fong-Fong, L., Morii, T. — 2009-09-12

Fluorescent biosensors based on the biological macromolecule are convenient tools for investigating the event occurring in the living cell. As for one of the candidates of such biosensors, we have reported a fluorescent sensor by utilizing a ribonucleopeptide (RNP) framework. Fluorescent RNP sensors are obtained from the fluorescent RNP library constructed by the combination of the RNA subunit, a substrate recognition unit selected by in vitro selection, and a fluorophore-modified peptide subunit. By taking the advantage of the noncovalent nature of fluorescent RNP complexes, RNP sensors with desired optical sensing properties are selected in a high-throughput manner. However, the noncovalent nature of the fluorescent RNP sensor is not suitable for practical applications. We report here a strategy to generate stable covalently linked RNP sensors and demonstrate a multiple ligands sensing system by using the covalently linked RNP sensors to detect biologically active ligands.

Structural aspects for the function of ATP-binding ribonucleopeptide receptors

Nakano, S., Fukuda, M., Mashima, T., Katahira, M., Morii, T. — 2009-09-12

We describe here analyses of the secondary structure of ATP-binding ribonucleopeptide (RNP) receptors. Mapping of the RNA structure of ATP-binding RNP receptors by using hydrolytic enzymes, chemical probing with dimethyl sulfate (DMS), and in-line probing indicated that ATP-binding RNP receptors take the loop structure at the nucleotide position of the "variable region". In addition, it was evident that a part of the consensus region located next to the variable region directly participated in the binding to ATP. The completely preserved three U nucleotides were essential for the binding of RNP to ATP as revealed by the affinity evaluation and the secondary structure analyses of the U nucleotides mutants of the ATP-binding RNP receptor. Interestingly, two mutants with an adenosine introduced to either of the two U nucleotides showed similar secondary structures to the original ATP-binding RNP. These results imply the possibility that the adenine base introduced at the U position acts just like the substrate ATP, and suggest that the U nucleotides in these positions interact directly to ATP.

Aptazyme-based biosensors using a eukaryotic cell-free translation system

Ogawa, A. — 2009-09-12

I have constructed a novel aptazyme-based biosensor system for detecting cofactors of the aptazymes using a cell-free luciferase synthesis in wheat germ extract. In this system, the activity of the aptazyme that is fused to a 5'-untranslated region of a luciferase gene can be detected as luciferase expression. In translating the aptazyme-fused mRNA as-is using a wheat germ cell-free translation system, the luciferase is almost not expressed because of the following triple suppression effects: (1) 5'-terminal three bases and (2) 5'-terminal duplex prevent the ribosome from binding to own mRNA; (3) if the ribosome binds, translation of a mimic gene in the aptazyme inhibits that of the downstream luciferase gene (OFF state). In contrast, in the presence of the aptazyme cofactor, the aptazyme in mRNA is self-cleaved to produce an aptazyme-free luciferase gene, which is translated efficiently (ON state). The ON/OFF efficiency and the detection limit of the aptazyme-based biosensor for theophylline are much higher and lower, respectively, compared to those of previously-reported one that utilizes a prokaryotic translation system.

The highly stabilized ribosome display selection of metal binding peptide aptamers

Wada, A., Ito, Y. — 2009-09-12

Development of methods for in vitro selection of peptide aptamers with target binding affinities have been expected to construct biocatalysts, biosensors, and molecular targeted drugs in leading-edge fields of biotechnology and medical therapies. Therefore, a highly stabilized ribosome display method has been devised for efficient selection of various peptide aptamers from an artificial peptide library (APL). This ribosome display selection is performed by using a ribosomal conjugate consisted of APL, Cv RNA-associating protein (Cvap), and mRNA having Cv RNA motif at 5’ terminus. The conjugate generated by in vitro translation can automatically link a peptide as phenotype and its mRNA as genotype, which can be stabilized by a high affinity association between Cv motif and Cvap. Here, in order to demonstrate the utility of this ribosome display method, we performed in vitro selection of peptide aptamers against a metal complex, and succeeded in identification and characterization of peptide aptamers with specific metal binding affinity. These results validate the concept to design APL in DNA level and indicate the versatility of the highly stabilized ribosome display selection.

Characterization of RNA aptamers against SRP19 protein having sequences different from SRP RNA

2009-09-12

SELEX is a conventional method to obtain high affinity nucleic acids to target molecules. In this study, high affinity RNA molecules against SRP19 protein were selected by using a randomized library. The primary and predicted secondary structures of the aptamers are different from those of S-domain RNA which is the natural target of SRP19 protein. Comparison of structural features between S-domain RNA and aptamers might enhance our understanding on RNA-protein interaction.

Structural analysis of ribonucleopeptide aptamer against ATP

2009-09-12

A ribonucleopeptide aptamer against ATP was obtained by the in vitro selection method. This ribonucleopeptide aptamer comprises a randomized and selected RNA linked to the Rev-responsive element (RRE) in complex with a peptide derived from an HIV Rev protein. The ribonucleopeptide aptamer selectively binds ATP in the presence of the Rev-derived peptide, exclusively. Here, we present the structural analysis of the ribonucleopeptide aptamer with NMR. The secondary structure of the RNA part of the aptamer, the selected RNA region linked to RRE, in the presence of the Rev-derived peptide was determined in an Ado-bound form. G:A and G:G base pairs, together with canonical base pairs, are formed in a duplex of RRE. The selected RNA region plays a crucial role in target binding. It has been found that the two U residues located in the selected RNA region trap Ado through the formation of the U:A:U base triple. This was directly confirmed by the HNN-COSY experiment through the detection of spin-spin couplings across the hydrogen bonds for Watson-Crick and Hoogsteen A:U base pairs in the U:A:U base triple.

NMR studies of HAC1 mRNA

Kawahara, I., Haruta, K., Kojima, C., Tanaka, Y. — 2009-09-12

HAC1 is a transcription factor related to Unfolded Protein Response (UPR) signaling in yeast. Processing of HAC1 mRNA on Endoplasmic reticulum (ER) plays a key role in UPR signaling pathway, but the recognition mechanism of HAC1 mRNA by processing enzyme Ire1p is still unclear. Here, the solution structure of HAC1 mRNA was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy, focusing on the structure of the recognition site of Ire1p in HAC1 mRNA. From the NOESY spectrum, imino proton signals of 5' processing regions of HAC1 mRNA were assigned and it was found that this region forms the stem-loop structure.

An isostructural G-G to A-A substitution within the HIV RRE RNA switches the specificity towards arginine-rich peptides

2009-09-12

The HIV Rev protein utilizes a short -helical arginine-rich RNA-binding domain to bind deeply within the major groove of an internal loop region of the Rev-response element (RRE) RNA. A G48-G71 base-pair which covaries to an isostructural A48-A71 base pair has been shown to play an important structure role in Rev-RRE binding. On the other hand, a high affinity RRE-binding peptide aptamer, the K1 peptide, was shown to have low binding affinity towards the RRE A48-A71 mutant, suggesting that the K1 peptide was recognizing the G48-G71 base-pair. In this study, in an attempt to understand the basis for the recognition of the G48-G71 base-pair by the K1 peptide, the selection of peptides that bind to the RRE A48A71 (RREAA) mutant was carried out. As a result, a peptide specific for the mutant, the LDN1 peptide, was identified. The LDN1 peptide was found to bind to the internal loop region of the RREAA, as in the case of the K1-RRE interaction. However, amino acids important for LDN1-binding to RREAA, were found to be distinct from those important for K1-binding to the RRE. These results demonstrate how subtle changes in RNA structure can dramatically alter the peptide-binding specificity of an RNA.

Docking simulation of polyamines on a kissing-loop RNA dimer

Imai, M., Chikatsu, D., Inomata, E., Oshima, T., Kawai, G. — 2009-09-12

Polyamines, especially branched polyamines such as tetrakis(3-aminopropyl)ammonium (Taa), stabilize the tertiary structure of RNA molecules.

In this study, we examined the polyamine binding site of the HIV-1 dimerization initiation site (DIS) in the kissing-loop dimer by the docking simulation. It was found that Taa binds predominantly to the kissing loop interaction site of DIS.

Non-enzymatic in vitro production of circular hammerhead ribozyme targeting the template region of human telomerase RNA

Ochi, A., Umekage, S., Kikuchi, Y. — 2009-09-12

Circular RNA is more stable than linear RNA both in vitro and in vivo because of its inaccessibility to exoribonucleases. Therefore, circularization of functional RNAs is a potentially useful methodology for designing therapeutic RNA reagents. We designed a circular hammerhead ribozyme that can cleave the template region of human telomerase RNA. This circular hammerhead ribozyme was generated by in vitro transcription followed by spontaneous self-circularization activity using the permuted intron-exon (PIE) method. Two-dimensional gel electrophoresis and alkaline digestion of the in vitro transcription products revealed that the circular hammerhead ribozyme could be produced by the PIE method. The purified circular hammerhead ribozyme cleaved the template region of human telomerase RNA in a magnesium-dependent manner. These results indicated that the circular hammerhead ribozyme generated by the PIE method maintained the specific feature of canonical hammerhead catalytic activity.

Preparations of hammerhead ribozymes for investigations of their cleavable sequences

2009-09-12

Recently, in hammerhead ribozymes, newly identified loop-loop interaction was found to be important for their activation. Therefore, we chemically synthesized a hammerhead ribozyme with this extra loop sequences and its mutant ribozymes, as well as their substrate RNA strands in order to clarify their cleavable sequences. After purification with an anion exchange column chromatography, we were able to obtain 44mer and 20mer RNA.

Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease

Mineta, Y., Okamoto, T., Takenaka, K., Doi, N., Aoyama, Y., Sera, T. — 2009-09-12

To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.

Construction of plants resistant to TYLCV by using artificial zinc-finger proteins

2009-09-12

Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato. An AZP was designed to block binding of the TYLCV replication protein (Rep) to the replication origin. The designed AZP had much higher affinities towards the replication origin than did the Rep, and efficiently blocked Rep binding in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. The current status of the construction of the AZP-expressing transgenic plants will be reported.

Thymidylate kinase: The lost chemotherapeutic target

Kandeel, M., Kato, A., Kitamura, Y., Kitade, Y. — 2009-09-12

Here we highlight the unusual substrate specificity of Plasmodium falciparum thymidylate kinase (PfTMK) and the validity of the enzyme as a new drug target. Furthermore, we predict that the Anaplasma marginale enzyme has attractive domain constituents and may be functionally different from other TMPKs. We postulate that thymidylate kinases could have multiple attractive functions in pathogens and may be a new drug target against numerous microorganisms.

Inhibition of influenza virus infection by targeting genome conserved region with non-natural nucleic acid

2009-09-12

Two highly conserved 15 base sequences of influenza A virus genome were identified by CONSERV software, which can detect contiguous conserved sequences of biological sequences. Antiviral effect of phosphorothioate oligonucleotide that target these conserved sequences was evaluated by plaque formation assay and cell viability assay. Pre-treatment of cells with anti-PB2 (RNA polymerase subunit) reduced the size of plaques in a sequence dependent manner. Post-treatment of cells with anti-PB2 phosphorothioate oligonucleotide inhibited the virus-induced cytotoxicity.

Inhibition of influenza virus by baculovirus-mediated shRNA

Suzuki, H., Saitoh, H., Suzuki, T., Takaku, H. — 2009-09-12

Influenza viruses A and B cause widespread infections of the human respiratory tract; however, existing vaccines and drug therapy are of limited value for their treatment. Here we show that bispecific short hairpin small-interfering RNA constructs containing an eight-nucleotide intervening spacer, targeted against influenza virus A or influenza virus B, can inhibit the production of both types of virus in infected cell lines. This multiple vector showed remarkable ability to cope with both influenza virus A or B. Furthermore, the Autographa californica multiple nuclear polyhedrosis virus can infect a range of mammalian cells, facilitating its use as a baculovirus vector for gene delivery into cells. In this study, baculovirus-mediated bispecific short-hairpin RNA expression markedly inhibited both influenza virus A and B production.

Regulation of vascular endothelial growth factor gene under hypoxia by using artificial transcription factors

Mori, T., Sasaki, J., Aoyama, Y., Sera, T. — 2009-09-12

The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To up- or downregulate the endogenous gene expression at will, we previously designed a 6-finger AZP to recognize a 19-bp target DNA in the VEGF-A gene, and fused the AZP with a nuclear localization signal and a transcriptional regulatory domain to generate artificial transcription factors (ATFs) for VEGF-A. Using the ATFs, we previously demonstrated efficient modulation of the endogenous VEGF-A expression under normoxia. In the present study, we examined whether these ATFs modulate the endogenous VEGF-A gene expression under hypoxic conditions as well. Enzyme-linked immunosorbent assays revealed that the gene-delivered ATFs up- or downregulated the VEGF-A expression efficiently under hypoxia.

Molecular mechanisms of apoptosis induced by 3'-ethynylcytidine

2009-09-12

1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)cytosine (3'-Ethynylcytidine; ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. We have investigated the cancer-cell death induced by ECyd, focusing on its molecular mechanisms. In ECyd-treated cells, RNase L is activated and involved in c-jun NH₂-terminal kinase (JNK) phosphorylation, followed by induction of mitochondria-dependent apoptosis. The mechanism of JNK phophorylation by RNase L was unknown. To investigate the mechanism, we performed the identification of RNase L-binding partners by proteomic approach using co-immunoprecipitation and mass spectrometry. We found that RNase L was associated with a protein (we named it Protein-190). At the same time, we observed that Protein-190 was amply phosphorylated. Furthermore, the participation of Protein-190 in the ECyd-induced apoptosis was supported by a knockdown experiment using small interfering RNA (siRNA). Thus, the number of ECyd-induced apoptotic cells was drastically decreased when Protein-190 was knocked-down. These results indicated Protein-190 as a regulator in apoptosis, and provide the possibility for a new clinical target in cancer chemotherapy.

Association of nuclear-intermediate filament lamin B1 with necrotic- and apoptotic-morphologies in cell death Induced by 5-fluoro-2'-deoxyuridine

2009-09-12

We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analyses. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Our present finding provides an interesting possibility that lamin-B1 may have an important role in regulating cell-death morphology.

Preparation of an ochre suppressor tRNA recognizing exclusively UAA codon by using the molecular surgery technique

Yokogawa, T., Hassan, H. M. B. C., Yokota, Y., Ohno, S., Nishikawa, K. — 2009-09-12

In order to create an ochre suppressor tRNA which exclusively recognizes UAA codon, we replaced the G34 at the first position of yeast tRNA^Tyr[GA] anticodon with pseudouridine34 (34) by using the molecular surgery technique. This tRNA^Tyr[A] recognized only the UAA codon as expectedly, but tRNA^Tyr[UA] made as a control also behaved similarly. This result may suggest that U34 must be somehow modified to facilitate the wobble-pairing to G at the third position of codon.

Screening of amber suppressor tRNAs suitable to introduce nonnatural amino acids into proteins by real-time monitoring of cell-free translation

Iijima, I., Hohsaka, T. — 2009-09-12

Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber suppressor activity of the tRNAs was evaluated by incorporation of a fluorescent nonnatural amino acid and fluorescent SDS-PAGE analysis of cell-free translation products, though the SDS-PAGE was troublesome and time-consuming. In this research, we developed an alternative method for the screening of amber suppressor tRNAs by real-time measurement of fluorescence resonance energy transfer (FRET) from GFP to BODIPY558-linked nonnatural amino acid, which was incorporated into the N-terminus of GFP by amber suppressor tRNAs. Using this method, we demonstrated that the screening of the amber suppressor activity of various prokaryotic Trp tRNAs was performed in a high-throughput manner.

Detection of enzymatic activities related to the tRNA splicing pathway in Methanosarcina acetivorans cell extract

2009-09-12

At least two separate enzymes, an endonuclease and a ligase, are thought to be involved in the tRNA splicing pathway. The yeast and archaeal endonucleases acting in the first step of tRNA splicing commonly produce 2', 3'-cyclic phosphate and 5' hydroxy group at the exon-intron borders. Despite this similarity in the first step of tRNA splicing, the subsequent mechanism of archaeal splicing pathway has not been elucidated yet. We have been searching for the archaeal ligase activitiy from Methanosarcina acetivorans. Here, we report the distinct activity of a splicing endonuclease detected in its cell extract.

Precise analysis of modification status at various stage of tRNA maturation in Saccharomyces cerevisiae

Ohira, T., Miyauchi, K., Sakaguchi, Y., Suzuki, T., Suzuki, T. — 2009-09-12

Transfer RNAs (tRNAs) are decorated with various post-transcriptional modifications which are enzymatically introduced at various stages of maturation. It is known that eukaryotic tRNAs are modified both in nucleus and cytoplasm. However, the order of tRNA modifications remains to be investigated. To unveil the precise timing of each modification associated with tRNA processing, we isolated precursor forms of Saccharomyces cerevisiae tRNAs at different stages of maturation, and analyzed their primary structures including modifications by mass spectrometry. The primary transcript of tRNA^Ile was isolated from the tRNA fraction co-precipitated with Lhp1p, a yeast homolog of La protein. The precursor tRNA^Ile without 3'-trailer sequence was isolated from the expression controllable strain when RNase P was transiently inactivated. Mass spectrometric analyses of these tRNAs revealed that many modifications were fully introduced at the stage of primary transcript, however, some modifications were partially introduced.

Production of yeast (m2G10) methyltransferase (Trm11 and Trm112 complex) in a wheat germ cell-free translation system

Okada, K., Muneyoshi, Y., Endo, Y., Hori, H. — 2009-09-12

Transfer RNA (guanine-N²-)–methyltransferase [tRNA (m²G10) methyltransferase] catalyzes a methyl-transfer from S-adenosyl-l-methionine to N²-atom of guanine at position 10 (G10) in tRNA and generates N²-methylguanine at position 10 (m²G10). Yeast enzyme contains two protein subunits (Trm11 and Trm112). Trm11 protein is expected to be a catalytic subunit and Trm112 contains a Zinc-finger. In yeast cells, Trm112 binds not only to Trm11 but also to other proteins such as Lys9, Trm9, and Mtq2. Therefore, the Trm112 protein may regulate population of several protein complexes. To address these issues, we started the study on synthesis of Trm112 related protein complexes. In this meeting, we report synthesis of active Trm11-Trm112 complex in a wheat germ cell-free translation system.

Trans-translation by tmRNA and SmpB

Une, M., Kurita, D., Muto, A., Himeno, H. — 2009-09-12

tmRNA has a dual function both as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch translation from a truncated or other problematic mRNA to the tmRNA's tag-encoding sequence. During trans-translation, tmRNA enters the ribosomal A-site without a codon-anticodon interaction, but with an SmpB which is a tmRNA binding protein. To further study the interaction of SmpB with ribosome and tmRNA, we have performed directed hydroxyl radical probing. It revealed that there are two SmpB binding sites at the A-site and P-site, and their C-terminal regions reside along the mRNA path in the 30S subunit. From these results, we propose a new type of molecular mimicry, in which both tmRNA and SmpB mimic the structures and functions of tRNA and mRNA during trans-translation, addressing how tmRNA preferentially recognizes the stalled ribosome, and what substitutes for a codon-anticodon interaction.

Physiological role of RsgA in ribosome biosynthesis

Hase, Y., Yokoyama, S., Kimura, T., Goto, S., Muto, A., Himeno, H. — 2009-09-12

RsgA is a unique GTP hydrolytic protein, in which the GTPase activity is significantly enhanced by the small ribosomal subunit. Depletion of RsgA causes slow cell growth as well as defects in the subunit assembly of the ribosome and the 16S rRNA processing, suggesting its involvement in the maturation of the small subunit. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA. Our recent study using chemical modification indicates that the binding of RsgA induces conformational changes around the A site, P site, and helix 44. These results suggest that RsgA is involved in the maturation step of the decoding center of the small subunit of ribosome. Here, we also show a physiological role of RsgA under stress condition.

Incipient complex formation between AP endonucleases and DNA containing AP site: A vital role of the tryptophan residue

Yamada, Y., Kodera, T., Ohishi, K., Kaneda, K., Shida, T. — 2009-09-12

To elucidate whether the tryptophan residues in the vicinity of the catalytic site are involved in AP site recognition and are critical for AP endonuclease activity, the AP endonucleases of the four subtypes in the ExoIII AP endonuclease family were characterized and compared the positions of the tryptophan residues. The positions of the catalytic amino acid residues, corresponding to Glu-34, Asp-229, and His-259 of ExoIII, are strictly conserved. On the other hand, the positions of the tryptophan residues, which are critical to the incipient complex formation, do not exist at a fixed position. There are four patterns at the position of the essential tryptophan residue.

Role of exposed aromatic residues in substrate-binding of CBM family 5 chitin-binding domain of alkaline chitinase

Uni, F., Lee, S., Yatsunami, R., Fukui, T., Nakamura, S. — 2009-09-12

Chitinase J (ChiJ) from alkaliphilic Bacillus sp. strain J813 has a multidomain structure containing a catalytic domain (CatD), a fibronectin type III like domain (FnIIID) and a chitin-binding domain (ChBD). It has been shown that the ChBD binds to an insoluble chitin and enhances its degradation by the CatD. Further binding study of the ChBD was performed with a glutathione-S-transferase fusion protein. This fusion protein showed binding abilities to insoluble chitin and chitosan. Two surface-exposed aromatic residues (Trp541 and Trp542) were found in the tertiary-structure model of ChBD and targeted for mutational analysis. Single and double mutations of the two aromatic residues decreased the chitin- and chitosan-binding abilities. It was revealed that these residues would be important for substrate-binding of the ChBD.

Expression behavior of high-pressure-compacted plasmid DNA in mammalian cell

Kimura, T., Konno, H., Fujisato, T., Kishida, A. — 2009-09-12

We have been developing a novel compaction method of plasmid DNA using high pressure technology, and previously found that the size of the plasmid DNA was decreased with increasing the pressurizing-strength and time. In the present study, we investigated the tertiary structural change and the expression behavior of the pressure-compacted plasmid DNA in cell in vitro. When the pressure-compacted plasmid DNA was reacted with restriction enzyme (EcoRI), a large amount of the EcoRI was required to cleave the pressure-compacted plasmid DNA than the non-pressurized plasmid DNA, suggesting that the structural change of plasmid DNA was induced by the pressurization. The expression of the pressure-compacted plasmid DNA injected into cells using microinjection method was analyzed. It was clear that the pressure-compacted plasmid DNA could effectively delay gene expression, suggesting that the controlled compaction of plasmid DNA by high pressurization would be regulate the transgene expression.

A novel convenient method for high bacteriophage titer assay

Yoshida, K., Nasu, Y., Shitami, N., Toyoda, H., Takemura, H., Oomori, K. — 2009-09-12

The recent various applications of phages (bacteriophages) including phage therapy have brought about a revival of phage investigation. The phage titer assay is indispensable for phage experiments. However, the conventional standard method is a plaque counting method which requires a little skill with tedious repeating operation. Furthermore, it is not directly applicable to high phage titers. In this paper, we describe a novel convenient "cross streak and paper disk assay method" for high titer concentration without plaque counting.

Author index

2009-09-12

Structural Biology of Riboswitch-mediated Gene Regulation and Argonaute-mediated Gene Silencing

Patel, D. J. — 2008-09-07

Characterization and Use of Tricyclic Fluorescent Nucleic Acid Base Analogues

2008-09-07

The two recently developed nucleic acid probe molecules tC and tC^O both have unique properties compared to other molecules in the family of fluorescent base analogues.^1–5 These tricyclic base analogues both form very stable base pairs with guanine and give minimal perturbations to the native structure of DNA.² We have found that tC^O is the brightest fluorescent base analogue reported⁴ and that tC also is very bright and has a fluorescence quantum yield that is virtually insensitive to its surrounding microenvironment within the nucleic acid³. These base analogues have so far been used in FRET-studies of a DNA-polymerase system⁶ and in initial anisotropy-studies of DNA-containing systems⁴.

Nucleic acid duplexes with zippers of additional nucleobases and aromatics in minor or major groove

2008-09-07

Two series of thymidine derivatives with additional nucleobases/aromatics attached to either the 5'(S)-C- or the 5-position were prepared by epoxide opening and/or "click chemistry" cycloaddition protocols and introduced into DNA duplexes. Interstrand base-base communication in the minor groove and intrastrand stacking interactions in the major groove were detected.

DNA and RNA Quadruplex ligands

2008-09-07

Guanine-rich nucleic acids can adopt unusual structures called guanine quadruplexes (G4) based on stacked guanine quartets. Both RNA and DNA backbones are compatible with G4 formation. As RNA and DNA quadruplexes may be recognized by ligands, it is important to understand the rules that govern the stability and specificity of these complexes. We explore the binding of a pyridine dicarboxamide derivative to various oligoribo- and oligodeoxyribo-nucleotides.

G-quadruplex forming oligonucleotides as finely tunable aptamers: towards better DNA mimics

Montesarchio, D. — 2008-09-07

The intense search for oligonucleotides (ODNs) endowed with pharmacological activities has led, in the past decade, to the identification of tens of candidate drugs, now being evaluated in preclinical or clinical trials. Based on G-rich DNA sequences, several aptamers, adopting G-quadruplex structures with different topologies, have been selected as potent in vitro antiviral and/or antitumoral agents.

In order to develop novel therapeutically relevant Gquadruplex-based aptamers, we have investigated - as a model compound – the ^5'd(TGGGAG)^3' sequence, known to be anti-HIV-1 active if 5'-modified with bulky aromatic residues. A set of 5'-conjugated analogues has been analyzed by integrated CD, DSC and molecular modelling studies, allowing a detailed biophysical characterization of the resulting G-quadruplexes. Following the assumption that the kinetically and thermodynamically favoured formation of the quadruplex complexes is a pre-requisite for their efficient antiviral activity, novel hybrid ODNs, carrying diverse terminal modifications, were prepared via a fully automated, on-line phosphoramidite-based strategy and evaluated for anti-HIV activity.

The use of light to investigate and modulate DNA and RNA conformations

Sen, D. — 2008-09-07

We report the use of light for two distinct kinds of investigation of the conformational changes of, respectively, DNA and RNA. First, ‘Deoxyribosensors’ are a class of DNA constructs that incorporate aptamers, and which report the binding of a ligand to the aptamer by attenuating the conformational/stacking relationship of two constituent DNA double helices within the sensor. Such attenuations can be monitored as electrical outputs via a light irradiation protocol. Second, RNA aptamers were selected for the specific binding of one but not the other isomer of different photochromic switch compounds. One such RNA aptamer, specific for binding the ‘closed’ isomer of a dihydropyrene compound, was used to create a highly effective, light-sensitive, RNA-cleaving ribozyme. Such ribozymes and riboswitches should find broad utility for the light-mediated control of gene expression within living cells and organisms.

Manipulation of the peptide-binding specificity of an RNA in a rational manner by combinations of specificity-altering mutations

Harada, K., Sugaya, M., Nishimura, F., Katoh, A. — 2008-09-07

In this study, the potential to manipulate the peptide-binding specificity of an RNA in a rational manner was investigated. First, variants of the Rev-response element (RRE) RNA with different specificities towards the natural binding partner, Rev, and two RRE-binding aptamers, the RSG-1.2 and K1 peptides, were identified. Next, hybrid RRE mutants with combinations of two sets of specificity-altering substitutions were tested for peptide-binding specificity. It was shown that, in most cases, the results of the combination of individual mutations were of an additive nature, therefore providing a way to manipulate the peptide-binding specificity of an RNA in a predictable manner.

Quantitative, Sensitive Analysis of DNA and RNA

Cantor, C. R. — 2008-09-07

DNA or RNA sequences that contain useful information about disease risk, disease occurrence, therapeutic response, and probable prognosis are potentially valuable biomarkers. They can be accessed by biopsy, or in ideal cases non-invasively from easily accessible fluids like blood or urine. Tolls are available for biomarker discovery, validation, and clinical use. Discovery usually requires whole genome analysis, and currently this is done with either nucleic acid arrays (DNA chips) or sequencing. Validation requires high quality data scalability to large numbers of samples. Clinical utility normally needs a high degree of automation, and for non-invasive approaches, great experimental sensitivity and specificity but on small numbers of samples.

No one platform can efficiently span the broad range of requirements and project sizes. SEQUENOM uses an automated mass spectrometry platform for the quantitative analysis of DNA and RNA in a variety of settings including genotyping, genecopy number measurements, gene expression, epigenetics, and automated bacterial and viral identification.

In collaboration with Amit Meller at Boston University, SEQUENOM is developing optically detected nanospores as a companion platform for its mass spectrometry offering. Both platform use similar homogeneous solution biochemistry to prepare samples. Both platforms depend on vary rapid digital signal processing for real time data analysis and interpretation. The nanopore method uses pores large enough to pass single-stranded DNA but too small to pass double strands. Hence pore passage strips off one of the DNA stands, and optical method are used to detect changes in fluorophores on this strand or attached to reporter probes, as it is stripped. We expect that, when mature, the nanopore method will be extremely const effective for whole genome analysis of genotypes, gene expression, epigenetics, and whole genome sequencing. A key aspect of the nanopore method is its speed. Post sample preparation, many analyses may require only seconds of instrument time.

Artificial molecular switches made from DNA

Friedrichs, E., Tsokou, A., Jungmann, R., Simmel, F. — 2008-09-07

The unique biochemical and biophysical properties of DNA can be utilized to construct nanoscale machines and switches, among them devices which can stretch and rotate, translocate, or perform computations. Switchable devices based on aptamers can controllably bind or release enzymes, which can be used to control biochemical reactions. DNA can also be used as a component for switchable materials, e.g. for the realization of switchable gels. These might find applications in the controlled release of particles or substances. An exciting possibility lies in the interaction of DNA nanodevices with RNA molecules. For instance, the behavior of DNA nanodevices may be controlled by natural or artificial gene regulatory mechanisms.

Construction of a photo-switchable gene for turning on and off gene expression with light irradiation

Liang, X., Fujioka, K., Tsuda, Y., Wakuda, R., Asanuma, H. — 2008-09-07

A photoresponsive GFP gene was constructed by attaching a T7 promoter that involves two azobenzene moieties as the photoswitch. The azobenzene moieties tethered on D-threoninol were inserted precisely into the sequence of T7 promoter at two positions in the nontemplate strand. By using azobenzene-tethered DNA as one primer, azobenzene was attached to GFP gene after PCR amplification. However, a single-stranded overhang involving azobenzene was formed because primer extension stopped at the position of azobenzene moiety. Interestingly we found that oligonucleotide complementary to the overhang could be ligated by T4 DNA ligase at the stopped position, and the intact photoresponsive T7 promoter was attached onto GFP gene. Furthermore, the in vitro expression of the constructed photoresponsive GFP gene was successfully switched on and off with light irradiation.

DNA Controlled Assembly of Liposomes

Jakobsen, U., Simonsen, A. C., Vogel, S. — 2008-09-07

DNA-encoding of solid nanoparticles requires surfacechemistry, which is often tedious and not generally applicable. In the present study non-covalently attached DNA are used to assemble soft nanoparticles (liposomes) in solution. This process displays remarkably sharp thermal transitions from assembled to disassembled state for which reason this method allows easy and fast detection of polynucleotides (e.g. DNA or RNA), including single nucleotide polymorphisms as well as insertions and deletions.

Zinc finger protein-based detection system of PCR products for pathogen diagnosis

Osawa, Y., Motoki, H., Matsuo, T., Horiuchi, M., Sode, K., Ikebukuro, K. — 2008-09-07

A novel detection system of PCR products from bacterial genomes using Zinc finger proteins was developed. Zinc finger proteins are DNA-binding proteins that can bind to dsDNA with high affinity and specificity. Since Zinc finger proteins can directly detect PCR products and can double-check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to construct the detection system for three pathogen, Legionella pneumophila, Salmonella spp. and Influenza A virus using well-characterized Zinc finger proteins. As a result, we succeeded in detecting the PCR products from Legionella pneumophila, Salmonella spp. and Influenza A virus using Sp1 and Zif268. Therefore, this methodology can be applied to the detection of most pathogen using various Zinc finger proteins.

LNA(R) incorporated siRNAs exhibit lower off-target effects compared to 2'-OMethoxy in Cell Phenotypic Assays and Microarray Analysis

2008-09-07

Despite the promise of short interfering RNAs (siRNA), contending with off-target is a challenge for RNAi users. To alleviate these problems, we have developed locked nucleic acid (LNA^®) modified siRNAs and optimized performance using cellular phenotypic assays as well as microarray analysis. During development, we compared LNA^® and 2'OMethoxy (2'OMe) chemistries placed strategically throughout the siRNA molecule and found a novel pattern of LNA^® placement that greatly improved the specificity of the siRNA and reduced it's toxicity in culture while preserving the potency of the siRNA. The improvements in specificity made by LNA^®-modified siRNAs were developed and validated by measuring the phenotypic signatures in a high content cell-based screening assay as well as comparison of the level of differentially expressed genes observed in microarray analysis between modified and unmodified siRNAs. HT screening of a collection of genes demonstrated that the LNA^®-modified siRNAs exhibits the best overall rate to elicit the expected phenotype, reduced toxicity and achieved an improved coherence of phenotype compared to 2'OMe-modified or unmodified siRNAs.

Building Biologically Active Nucleic Acid Nanocomplexes

2008-09-07

The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.

Recognizing and Controlling Biomolecules with "Smart" Hybridization-based Switches

Seitz, O., Grossmann, T. N., Thurley, S., Roglin, L. — 2008-09-07

The rules that govern the formation of DNA duplex structures are well known. On one hand, this process is used in the design of probe molecules that report the presence of target nucleic acids by responding to changes of structure and reactivity. On the other hand, molecules may be developed that transduce changes of nucleic acid structure to changes of peptide structure, and vice versa. Applications in the fields of bioanalytical chemistry and synthetic biology are discussed.

PNA-peptide conjugates as intracellular gene control agents

2008-09-07

Serum-stabilized PNA-internalization peptides (Pip) conjugated to PNA complementary to the 705 aberrant β-globin splice site are able to correct splicing and increase luciferase production in Hela pLuc705 cells with sub µM EC₅₀ in the absence of a transfection agent. Inhibition of microRNA-122 in liver cells is achieved by treatment with complementary PNA containing just a few attached Lys residues, again without need of a transfection agent.

Structural and functional prerequisites for ribosomal nascentpeptide acceptors: Attempts to decipher the nature of the ribosome's catalysis of peptide bond formation.

Michel, B. Y., Krishnakumar, K. S., Johansson, M., Ehrenberg, M., Strazewski, P. — 2008-09-07

Aminoacyl ribonucleoside analogues that are capable of binding to the acceptor site of ribosomes and taking over the nascent peptide bear, if properly designed, thepotential of antibiotic and cytostatic activity. Here wepresent a study on the intrinsic conformations of natural and synthetic peptide acceptors and the basicities of their peptide accepting amino groups. The conformations and thermodynamic parameters of several synthetic puromycin analogues have been elucidated through ab intio calculations as well as temperature and pH dependent ¹H NMR experiments. The intrinsic basicities of their peptide accepting amino groups were determined through ¹H NMR and compared to the effective basici-ties of the peptide accepting amino groups of aminoacyl transfer RNAs with the same amino acid side chains, as estimated from the pH dependent kinetics of mRNAprogrammed ribosomal peptidyl transfer.

The acetal levulinyl ester (ALE) group for the 2'-hydroxyl protection of ribonucleosides and the synthesis of oligoribonucleotides

Lackey, J. G., Damha, M. J. — 2008-09-07

A novel 2'-O-ALE (Acetal Levulinyl Ester) uridine phosphoramidite derivative has been efficiently prepared and used for the solid-phase synthesis of a chimeric oligonucleotide strand, 5'-rU11-dT-3'. The average coupling yield of the phosphoramidite was comparable to one obtained with a 2'-TBDMS rU phosphoramidite reagent. Upon completion of the RNA chain assembly, the cyanoethyl phosphate protecting groups were removed using a solution of triethylamine in acetonitrile (2:3 v/v). The 2'-O-ALE protecting groups were cleaved under hydrazinolysis conditions. Finally, the RNA oligonucleotide was released from the Q-CPG support when treated with 1 molar TBAF in THF. When the TBAF step was carried out prior to hydrazinolysis, an oligonucleotide with its intact 2'-OALE groups was obtained.

Triplex glue by synthesizing conjugated flexible intercalators

2008-09-07

Bulge insertions of conjugated intercalators into the DNA triplex structure are found to give a dramatic contribution to the triplex stability. On the other hand insertions of conjugated intercalators are found to diminish quadruplex structures and in this way breaking down the self association of G-rich oligonucleotides under physiologically potassium ion conditions. A large number of intercalators are described here and they all result in dramatic increases of thermal stability of the corresponding triplexes. Another interesting aspect of conjugated intercalators is their use for assembling alternate strand triplexes. Targeting of neighbouring purine sequences on each their strand in the duplex DNA is a challenge for the 5'- 5' connectivity of the TFOs because of a large distance between the 5'-ends. The intercalator approach offers a linkage with the proper combination of flexibility and rigidity to produce alternate strand triplexes with higher stability than a similar wild type triplex of the same total length.

New strategies for the synthesis of unmodified and modified oligonucleotides

Sekine, M., Ohkubo, A., Seio, K. — 2008-09-07

In this paper, our recent studies on the synthesis of unmodified and modified oligonucleotides are comprehensively reviewed. We have developed a new synthetic strategy using TrS, a new 5'-protecting group, that enabled us to reduce one of the steps previously required for DNA synthesis. The "activated phosphite method" without base protection could provide a new tool for the synthesis of DNA oligomers involving baselabile functional groups such as N-acylated nucleobases capable of Watson-Crick base pairing. This new method was successfully applied to the synthesis of oligodeoxynucleotides incorporating cytosine N-oxide or adenine N-oxide that could not be synthesized by the current methods. Furthermore, several approaches we recently developed for the synthesis of several kinds of 2'-O-modified RNA oligomers will be reported.

RNA interference in silencing of genes of Alzheimer's disease in cellular and rat brain models

2008-09-07

Accumulation of insoluble aggregates of β-amyloid peptide, a cleavage product of amyloid precursor protein, is thought to be a central step in the pathogenesis of Alzheimer's disease. The major enzymes required for the generation of toxic amyloidbeta peptide are β- (BACE1) and -secretases. Here, we present the rational design and the application of synthetic and lentivirus vector-encoded siRNAs for specific and efficient knockdown of overexpressed and endogenous BACE1, both in dividing and neural stem cells and in a rat brain. We also tested an approach to anti-amyloid therapy by the use of the allele-specific siRNAs to silence the mutant presenilin 1 (L392V PS-1), the main component of -secretase, responsible for development of Familial Alzheimer's disease. Reducing the level of β-amyloid accumulation in the brain could be beneficial for metabolic studies as well as potential therapeutic approach for prevention and treatment of Alzheimer's disease.

Synthesis of the new nucleoside analogue connecting 2-amino-6-vinylpurine to the 2'-deoxyribose skeleton via the methylene linker

Kurose, Y., Taniguchi, Y., Nagatsugi, F., Sasaki, S. — 2008-09-07

We have previously reported that the 2-amino-6- vinylpurine nucleoside exhibits the highly efficient and selective cross-linking reaction toward the cytosine base at the target site in the duplex DNA. The nucleoside analogues that connect the 2-amino-6-vinylpurine to the 2'-deoxyribose skeleton through the ethylene or the butylene linker formed the cross-link selectively to the adenine base of the TA pair or the cytosine base of the GC pair in the triplex DNA, respectively. They did not form cross-link in the duplex DNA. These results lead us to study in detail the relationship between the linker length and the cross-linking ability. In this study, we describe the synthesis of the new nucleoside analogue that connects 2-amino-6-vinylpurine to the 2'- deoxyribose unit via the methylene linker.

Potent single stranded RNA inhibition

Koch, T. — 2008-09-07

The high affinity provided by LNA is the basis for its high antisense potency. This property offers furthermore the opportunity to make shorter than usual antisense oligonucleotides exhibiting very high in vivo potency and efficacy. The potency of short-stranded LNA is comparable to the very best lipid formulated siRNA's. But in contrast to siRNA short-stranded LNA provides a common platform for highly potent mRNA and microRNA inhibitors.

All these features of LNA will be comprehensively illustrated in the presentation. Inhibition of coding RNA will be illustrated using ApoB-100 as the target, and inhibition of miR-122 will be used to illustrate inhibition of non-coding RNA. The conclusions in the presentation are all based on comprehensive rodent and non-human primate data.

Click chemistry and Oligonucleotides: How a simple reaction can do so much

2008-09-07

Copper catalyzed Alkyne Azide 1,3-dipolar cycloaddition (CuAAC) "click reaction" was applied for the construction of oligonucleotide conjugates, circular objects and phosphodiester glyco-clusters. To this end, several strategies were developed to introduce either alkyne or azide functions into an oligonucleotide.

Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts

Tretyakova, N., Guza, R., Matter, B. — 2008-09-07

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (^MeC, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O⁶-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of ^MeC were prepared, and their effects on reactivity of base the paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N²-BPDEdG adducts.

A Whole Base-Labile Strategy for RNA Synthesis with 2'-O-acetalester Protections

Lavergne, T., Martin, A., Debart, F., Vasseur, J.-J. — 2008-09-07

The use of base-labile groups for 2'-OH protection is a major challenge for RNA synthesis. A new method with acyloxymethyl groups has been developed. These groups were fully compatible with standard base-labile protections for nucleobases and phosphates and were removed in a short two-step all-base deprotection. Oligoribonucleotides were synthesized efficiently, rapidly and in high purity without chain rupture or isomerisation via this new whole base-labile strategy.

Novel base-functionalized DNA. Efficient methodology for construction and bioanalytical applications

2008-09-07

A novel efficient two-step methodology for the construction of base-functionalized DNA is based on direct aqueous cross-coupling reactions of unprotected nucleoside triphosphates followed by polymerase incorporation. Preliminary applications of the modified DNA in electrochemical detection and bioanalysis are outlined.

Recent Developments in the Synthesis of RNA Oligonucleotides for Potential Therapeutic Applications.

Cieslak, J., Grajkowski, A., Ausin, C., Beaucage, S. L. — 2008-09-07

The 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl group for 2'-hydroxyl protection has been successfully employed in the solid-phase synthesis of AUCCGUAGCUAACGUCAUGG. The use of the 2'-Omethylthiomethyl (2'-O-MTM) protecting group in the development of a cost effective approach to RNA synthesis has been evaluated through the solid-phase synthesis of [2'-O-MTM U]19dT. Given the sensitivity of 2'-thioacetals to acidic conditions, the base-labile [2-(9- fluorenyl)propyl-2-oxy]carbonyl group for 5'-Oprotection of ribonucleosides has been designed to accommodate the synthesis of oligoribonucleotides functionalized with 2'-thioacetal groups.

Repair of DNA-protein crosslink damage: Coordinated actions of nucleotide excision repair and homologous recombination

Ide, H., Nakano, T., Salem, A. M.H., Terato, H., Pack, S. P., Makino, K. — 2008-09-07

DNA-protein crosslinks (DPCs) are extremely bulky DNA lesions, and steric hindrance imposed by covalently trapped proteins would hamper the transaction of DNA such as replication, transcription, and repair. However, it has been largely elusive how cells mitigate the genotoxic effect of DPCs. We have recently shown that nucleotide excision repair (NER) and homologous recombination (HR) differentially contribute to the repair of DPCs in E. coli cells. Several lines of genetic and biochemical evidence indicate that NER repairs DPCs with crosslinked proteins (CLPs) of sizes less than 12–14 kDa, whereas DPCs with oversized CLPs are processed exclusively by RecBCD-dependent HR. The present result shows that cells use the coordinated actions of NER and HR to deal with unusually bulky DNA lesions like DPCs.

Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing

Siomi, M. C., Siomi, H. — 2008-09-07

Small RNAs triggering RNA silencing are loaded onto Argonautes and then sequence-specifically guide them to target transcripts. Epitope-tagged human Argonautes (hAgo1, hAgo2, hAgo3, and hAgo4) are associated with siRNAs and miRNAs, but only epitope-tagged hAgo2 has been shown to have Slicer activity. Contrarily, how endogenous hAgos behave in respect to small RNA association and target RNA destruction has remained unclear. Recently, we produced monoclonal antibodies for individual hAgos and characterized small RNAs specifically associated with hAgo2 and hAgo3 endogenously expressed in Jurkat cells.

Physicochemical Stability of NOX-E36, a 40mer L-RNA (Spiegelmer) for Therapeutic Applications

Maasch, C., Buchner, K., Eulberg, D., Vonhoff, S., Klussmann, S. — 2008-09-07

Spiegelmers are structured mirror-image oligonucleotides that are designed to bind and inhibit pharmacologically relevant target molecules. The synthesis and purification of mirror-image oligonucleotides is comparable to the manufacturing of standard oligonucleotides that consist of naturally configured nucleotides. Due to the use of the nonnatural L-nucleotides in Spiegelmers, these oligonucleotides show an exceptional biostability. Further, they also display a high physicochemical stability in solution. These properties make them interesting substances for drug development.

Inhibition of picornaviruses by means of RNA interference

2008-09-07

Picornaviruses are a class of RNA viruses with a single-stranded genome in positive orientation. Since the prospects of treatment are limited, we employ RNA interference (RNAi) as an antiviral tool to inhibit different picornaviruses. We identified small interfering RNAs (siRNAs) against the 3D RNA dependent RNA polymerase of coxsackievirus B3 that were capable of efficiently inhibiting the virus. Targeting of the conserved 5' UTR of the virus turned out to be a challenging task since stable structures of this region are detrimental to silencing. We developed a rational strategy to solve this problem and found an siRNA containing locked nucleic acids (LNAs) to possess high antiviral potency. To analyse the mechanism of virus inhibition in more detail, LNAs were incorporated into the siRNA to inactivate either of the siRNA strands. These experiments clearly revealed that only the genomic plus-strand but not the intermediary synthesised minus-strand can be targeted by siRNAs. Furthermore, siRNAs were employed to silence the virus receptor on the host cell and thus prevent viral spread.

Molecular engineering of DNA bases: Building block for functional molecular probes in biomedicine

Wu, Y., Kim, Y., Tan, W. — 2008-09-07

Protein-Facilitated Ribozyme Folding and Catalysis

Zingler, N., Solem, A., Pyle, A. M. — 2008-09-07

In vivo, large RNAs rely on proteins to fold to their native conformation. In the case of the S. cerevisiae group II intron ai5, the DEAD-box protein Mss116 has been shown to promote the formation of the catalytically active structure. However, it is a matter of debate whether it does this by stabilizing on-pathway intermediates or by disrupting misfolded structures. Here we present the available experimental evidence to distinguish between those mechanisms and discuss the possible interpretations.

2'-O-Methoxyethyl/2'-Fluoro Modified Oligonucleotides Result in More Potent Inhibition of micro RNA-122 in Vivo: A Target implicatedin HCV Replication

2008-09-07

MicroRNAs are endogenous 20-24 nt small non-codingRNAs that have profound roles in multiple developmental and cellular processes. Dysregulation of microRNAs can lead to a host of pathologies suggesting that microRNAs could be important for therapeutic intervention in cancer, metabolic diseases, autoimmune disorders and viral diseases. Through recent studies,mir-122 has emerged as a potential target for metabolic diseases and HCY. Chemical modifications are essential to achieve clinically relevant potency and efficacy andtherapeutic index of anti-miRNA oligonucleotides. Wehave evaluated more than 65 chemically-modified ASOs for their ability to inhibit the activity of miR-122 inmice. Inhibition of miR-122 with ASOs resulted inincreased levels of miR-122 target gene mRNAs in theliver, as well as lowering of plasma cholesterol in a dose dependant manner. The current investigation led to the identification of a chimeric 2'Fluoro/2'-O-methoxyethyI(2'OME) modified motif with improved efficacy and 5-10 fold improvement in potency compared to LNA/DNAmodified and uniform 2'-MOE-PS compounds. Theseefforts have identified significantly improved anti-miR-122 ASOs for further evaluation as anti-HCV therapeutic agents.

An efficient fluorescent method for selective detection of mature miRNA species

Kato, Y. — 2008-09-07

Methods for specific detection of RNA molecules have been widely developed in biotechnology and diagnostic field. The diagnostic target is recently expanded to non-coding RNA, including microRNAs (miRNAs). miRNAs are initially synthesized as precursor, and then processed to their physiologically active species, miRNAs. Detection of only the active mature miRNA without interference of the precursor forms is extremely important for their functional and physiological significance. We developed a novel fluorescent DNA probe to detect the mature miRNA with high specificity. Since the probe discriminates the mature miRNA from the precursor without electrophoretic separation, it should be useful for real-time detection of the mature miRNAs.

In vitro selection of a DNAzyme with three modified nucleotides

Hollenstein, M., Hipolito, C., Lam, C., Perrin, D. — 2008-09-07

Three modified nucleosides have been used in the in vitro selection of the self-cleaving DNAzyme 9-33. This DNAzyme operates in the absence of divalent metal cations with a first-order constant of 0.05 min^–1.

Transition state analogues in quorum sensing and SAM recycling

2008-09-07

Transition state structures can be derived from kinetic isotope effects and computational chemistry. Molecular electrostatic potential maps of transition states serve as blueprints to guide synthesis of transition state analogue inhibitors of target enzymes. 5'- Methylthioadenosine phosphorylase (MTAP) functions in the polyamine pathway by recycling methylthioadenosine (MTA) and maintaining cellular Sadenosylmethionine (SAM). Its transition state structure was used to guide synthesis of MT-DADMe-ImmA, a picomolar inhibitor that shows anticancer effects against solid tumors. Biochemical and genomic analysis suggests that MTAP inhibition acts by altered DNA methylation and gene expression patterns. A related bacterial enzyme, 5'-methylthioadenosine nucleosidase (MTAN), functions in pathways of quorum sensing involving AI-1 and AI-2 molecules. Transition states have been solved for several bacterial MTANs and used to guide synthesis of powerful inhibitors with dissociation constants in the femtomolar to picomolar range. BuT-DADMe-ImmA blocks quorum sensing in Vibrio cholerae without changing bacterial growth rates. Transition state analogue inhibitors show promise as anticancer and antibacterial agents.

Chemistry and Structure-Activity Relationship of Antibacterial Nucleoside Natural Products

Ichikawa, S., Matsuda, A. — 2008-09-07

Synthetic methodology of the caprazamycins, which are promising antibacterial nucleoside natural products, was developed. Palmitoylcaprazol, which possesses a simple fatty acyl side chain at the diazepanone moiety of caprazamycins, has been synthesized and exhibited antibacterial activity against pathogens threatening a public health. Simplification of the caprazamycins was further pursued to develop diketopiperazine analogs.

Development of A3 Adenosine Receptor Ligands

Jeong, L. S. — 2008-09-07

4'-Thioadenosines have been discovered as novel templates for A₃ adenosine ligands. Among these, 4'-thioadenosine-5'-monoalkyluronamides were discovered as novel potent and selective A₃ adenosine receptor agonists, while 4'-thioadenosine-5'-dialkyluronamides and truncated 4'-thioadenosine derivatives exhibited potent and selective antagonism at the A₃ adenosine receptor.

Synthesis and properties of ({alpha}-P-borano)-nucleoside 5'-triphosphate analogues as potential antiviral agents

2008-09-07

The -P-borano modification, where one of the -phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the -P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl -P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions. These analogues will be used for future structure-activity relationship (SAR) studies.

Nucleoside Diphosphate Prodrugs

Meier, C., Jessen, H. J., Balzarini, J. — 2008-09-07

Nucleoside analogs are widely applied in antiviral and antitumor therapy. A severe limitation of these compounds arises from the need of biotransformation to the eventually active nucleoside triphosphates by stepwise addition of phosphate by kinases. This problem can be circumvented by employing reversibly masked nucleotides (prodrugs). However, the known concepts to bypass enzyme activation have almost exclusively been applied to nucleoside monophosphates. Here, we report on the transfer of the bis- (acyloxybenzyl)-concept (BAB-concept) from nucleoside monophosphates to nucleoside diphosphates (NDP) of the anti HIV drugs AZT and d4T. After successful synthesis and isolation of the compounds (BAB-NDPs), it was shown that these compounds exhibit promising hydrolytic properties ranging from high stability at physiological pH to very low stability in cellular extracts. Furthermore, we demonstrated that for some of the compounds a selective cleavage mechanism resulted in the exclusive delivery of the corresponding nucleoside diphosphate within 15 minutes without any degradation of the pyrophosphate unit, which is a unique result in the field of NDP-prodrugs.

Inhibition of Orotidine-5'-monophosphate decarboxylase - Discoveries and lessons

Kotra, L. P., Pai, E. F. — 2008-09-07

Orotidine-5'-monophosphate decarboxylase (ODCase) is one of most proficient enzymes, and this enzyme catalyzes the decarboxylation of orotidine-5'- monophosphate (OMP) to uridine-5'-monophosphate (UMP). A number of C6-substituted uridine derivatives are designed to investigate the mechanism of decarboxylation by this enzyme. In this process, novel reactions and mechanisms were uncovered for this decarboxylase. This led to the discovery of novel ODCase inhibitors and their biological activities. Medicinal chemistry of these novel inhibitors of ODCase in the context of its catalytic mechanism, and therapeutics development will be discussed.

Multifunctional envelope type nano device for non-viral gene delivery: Concept and application for nanomedicine

Harashima, H. — 2008-09-07

Preparation of Mannosylated Oligoribonucleotides

Zhao, Y., Yan, H. — 2008-09-07

Multivalent mannosides were synthesized for attachment to oligoribonucleotides via 3,4-diethoxy-3-cyclobutene-1,2-dione (squarate). The conjugates have potential to be used in receptor-mediated endocytosis of small interference RNAs into dendritic cells through interactions with mannose-binding lectins.

Tyrosine-modified PEI: A novel and highly efficient vector for siRNA delivery in mammalian cells

Creusat, G., Zuber, G. — 2008-09-07

Delivery of synthetic small interfering RNA (siRNA) into cell remains the major obstacle to its biological activity. Cationic polymers, and in particular the "proton sponge" polyethylenimine (PEI), has shown promise for cancer gene therapy but appears inefficient for siRNA delivery. Here we report that modifying branched PEI with amino acids led to efficient siRNA delivery into mammalian cell lines, even in the presence of serum, and at dose as low as 1 nM.

Designer DNA Architectures for Nanobiotechnology

Liu, Y., Yan, H. — 2008-09-07

DNA electron transfer mechanism and dynamics

Lewis, F. D. — 2008-09-07

The dynamics and efficiency of photoinduced charge separation in synthetic DNA hairpins and dumbbells possessing stilbenedicarboxamide (Sa) and stilbene-diether (Sd) linkers separated by short poly(dA)-poly(dT) base pair sequences have been investigated by means of time resolved fluorescence and transient absorption spectroscopy. Charge separation occurs via a single step superexchange mechanism at short distances and by a multistep hole transport mechanism when the linkers are separated by two or more base pairs. The dynamics and efficiency of hole transport are strongly distance dependent over the first few base pairs, but are relatively insensitive to distance at larger separations. The distance dependence is attributed to a Coulomb cage effect. Hole transport across alternating AT base sequences is less efficient than across poly(dA) sequences. Introduction of guanine into the poly(dA) sequence can either enhance or diminish the efficiency of charge separation, depending upon the location of G. Our experimental results are interpreted using several theoretical models.

Addressable Molecular Node Assembly - Functional DNA Nanostructures

Tumpane, J., Lundberg, E. P., Wilhelmsson, L. M., Brown, T., Norden, B. — 2008-09-07

The use of nucleic acids as a nanomaterial is becoming increasingly widespread due to the suitability of the hydrogen-bonding patterns and sequence specificity inherent to the double-helix. As minimisation of size becomes ever more important it is imperative to employ nucleic acids in the most efficient and functional manner possible. To this end we have constructed DNA nanostructures on what may be the smallest possible scale (basic components of just 10 bp) that not only reliably self-assemble but also where each unit of a 2-dimensional DNA network can be uniquely identified and selectively functionalised.^1,2.3 On this length scale and using full addressability of the network to engrave specific pathways on the scaffold, energy and electron transfer become efficient for potential information storage applications.⁴

Synthesis of 2',3'-Cyclohexene Bicyclic Nucleoside Analogues as Antiviral Compounds

2008-09-07

Chiral syntheses of a series of hexahydroisobenzofuran (HIBF) nucleosides have been accomplished via glycosylation of a stereo-defined (syn-isomer) sugar motif with the appropriate silylated bases. All nucleoside analogues were obtained in 52-71% yield as a mixture of - and β-anomeric products increasing the breadth of the novel nucleosides available for screening. Nucleoside derivatives were tested as inhibitor of HIV-1 in human peripheral blood mononuclear (PBM) cells.

Preparation of Nucleoside-Carbohydrate Phosphodiester Prodrug Analogues by Chemoenzymatic Procedure

Rodriguez-Perez, T., Fernandez, S., Sanghvi, Y. S., Goto, V., Ferrero, M. — 2008-09-07

An efficient synthesis protocol for the glucosylnucleoside phosphodiester derivatives has been developed. These mononucleotides were designed to act as pronucleotides with potential to deliver the parent compound as its monophosphate. Key step of the synthesis is the regioselective hydrolysis of peracetylated -D-glucose catalyzed by Candida rugosa lipase.

RNA Synthesis: Phosphoramidites for RNA synthesis in the reverse direction. Highly efficient synthesis and application to convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3'- end.

Srivastava, S. C., Pandey, D., Srivastava, N. P., Bajpai, S. P. — 2008-09-07

Defined sequence RNA synthesis by 3'->5' direction is now well established and currently in use for synthesis and development of vast variety of therapeutic grade RNA and Si RNA etc. A number of such synthetic RNA requires a modification or labeling of 3'- end of an oligonucleotide. The synthesis of 3'- end modified RNA requiring lipophilic, long chain ligands or chromophores, using 3' -> 5' synthesis methodology is challenging, requires corresponding solid support and generally results in low coupling efficiency and lower purity of the final oligonucleotide in general because of large amount of truncated sequences containing desired hydrophobic modification. We have approached this problem by developing reverse RNA monomer phosphoramidites for RNA synthesis in 5' -> 3'- direction. They lead to very clean oligonucleotide synthesis allowing for introduction of various modifications at the 3'- end.

Studies of sequence-specific recognition and interaction of bishairpin polyamide minor groove binders with target DNA duplexes

Boutorine, A.S., Buchmann, W., Halby, L., De Pauw, E. — 2008-09-07

The binding of bis-MGB to target DNA was studied by DNase footprint, native gel shift, circular dichroism, thermal dissociation, electrospray mass-spectrometry, and molecular modelling methods. A new method for the determination of the relative affinity of ligands against various dsDNA sequences was elaborated by using ESI-QTOF mass spectrometry. Information about affinity, sequence preferences, conformation and mode of interaction between bis-MGB and target DNA was obtained. Our experiments demonstrated that MGB have different affinity for similar cognate target sequences depending on the sequence context of the target region and other structural factors.

Binding of two bis-bipyridine minor groove binders to a DNA template in the presence of Cu2+ ions

Brazier, J. A., Onishi, I., Sasaki, S. — 2008-09-07

Some diseases are associated with abnormally extended regions of triplet repeats. These repeating regions are an attractive target for both diagnostic and therapeutic goals. In an attempt to approach to this goal, we have focused on establishment of an allosteric binding mechanism, in which the binding of the ligand promotes the next ligand binding. In the previous study, we already reported that the ligand having the bipyridine unit for binding with Cu²⁺ and the Hoechst33258 for binding to A₃T₃ site displayed Cu²⁺- mediated assembly on the DNA with two A₃T₃ sites. In this study, we synthesized the new ligands containing two bipyridine units attached to Hoechst33258 by different length linkers. It was expected that the bipyridine-Cu²⁺ complexation would enhance assembly of a number of the lingand on the DNA sequence with repeating regions. UV spectroscopy has been used to demonstrate the binding of these ligands to a DNA template mediated by the complexation of Cu²⁺ ions.

Recognition of Homopyrimidine Mismatches by Distance-Constrained Macrocyclic bisintercalators.

Bahr, M., Gabelica, V., Granzhan, A., Weinhold, E., Teulade-Fichou, M.-P. — 2008-09-07

Binding of three macrocyclic bisintercalators to mismatch-containing duplexes was analyzed by thermal denaturation experiments, electrospray mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bisintercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched one and affinity in the submicromolar range (Kd). The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch.

The interaction between the purine motif triplex and the triplex DNA-binding domain of Saccharomyces cerevisiae Stm1 protein

Katayama, T., Torigoe, H. — 2008-09-07

Saccharomyces cerevisiae Stm1 protein (273 amino acids) is a purine motif triplex DNA-binding protein. We have previously found that Stm(1-113) (amino acids 1-113) is the minimal domain to specifically bind with the purine motif triplex. Here, to reveal the triplex recognition mechanism of Stm(1-113), we have examined the interaction between Stm(1-113) and each of the purine motif triplexes with various lengths and base sequences. As the length of the target triplex was increased, the binding affinity of Stm(1-113) to the target triplex was increased. Stm(1-113) had the ability to bind to the purine motif triplexes with various base sequences. We conclude that Stm(1-113) may recognize the shape of the triplex rather than the base sequence of the triplex.

Effect of cationic comb-type copolymer on the B- Z transition of poly(dG-dC){middle dot}poly(dG-dC)

Shimada, N., Kano, A., Maruyama, A. — 2008-09-07

The structural transition of a double helical DNA from B-DNA to Z-DNA, B-Z transition, has been received growing attention because of its potential roles in biological systems and its applicability to nanobiotechnology. It was known that the B-Z transition is induced by cationic molecules. However, there are only a few reports describing cationic polymers as potential B-Z inducers. In this study, we demonstrated that cationic comb-type copolymer, poly(L-lysine)-graft-dextran, induced the B-Z transition in which distinct intermediate formation was involved. Furthermore, we suggested that the grafted-dextran plays an important role in the B-Z transition.

A pteridine derivative with electron-withdrawing groups for binding and sensing of nucleobases in AP site-containing DNA duplexes

Kanai, E., Nishizawa, S., Teramae, N. — 2008-09-07

A pteridine derivative having electron-withdrawing CF₃ groups, 2-amino-6,7-bis(trifluoromethyl)-4-hydoroxypteridine (2CF₃-pteridine), is presented as a candidate for multi-functional fluorescent ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 8.0 (I = 0.1 M, at 5°C), 2CF₃- pteridine can bind to guanine, cytosine and thymine opposite an abasic site in DNA duplexes (5'-TCTGC GTCCA GXG CAACGCACAC-3'/3'-AGACG CAGGT CNC GTTGCGTGTG-5', X = abasic site; Spacer-C3, N = G, C, A, T). For these three nucleobases, the binding of 2CF₃-pteridine is explained by 1:1 complexation, and the binding affinities are comparable (K₁₁ / 10⁵ M^-1: G: 3.0; C: 1.6; T: 3.3). Binding-induced fluorescence responses are effectively different between guanine and pyrimidines (C, T): the binding to pyrimidines is accompanied by a significant change in the shape of fluorescence spectra. These binding and sensing properties allow a detection of G/T or G/C mutation based on a single fluorescence ligand.

Effect of an alkyl amino group on the binding of 1,8-naphthyridines to AP site-containing DNA duplexes

Ichihashi, T., Sato, Y., Seino, T., Nishizawa, S., Teramae, N. — 2008-09-07

A 1,8-naphthyridine derivative having a positively charged side-chain, N-(3-aminopropyl)-5,6,7-trimethyl-1,8-naphthyridin-2-amine (APATMND), is synthesized, and its binding to AP site-containing DNA duplexes (5'- GCA GCT CCC GXG GTC TCC TCG-3'/ 5'-CGA GGA GAC CNC GGG AGC TGC-3', X = AP site; dSpacer, N = C, T) is examined in solutions buffered to pH 7.0 (I = 0.11 M, at 20°C). Fluorescence titration experiments reveal that, as compared to a parent ligand, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), capable of selectively binding C over T opposite an AP site in the duplex (K_d/nM: C: 56, T: 100), APATMND shows a stronger binding affinity for T, while an affinity for C is reduced (K_d/nM: C: 135, T: 37). An examination of salt dependence of binding constants reveals that a polyelectrolyte contribution (G_pe) is indeed increased for C- and T-bindings of APATMND, but a loss of non-polyelectrolyte contribution (G_t) is significant when binding to C. These binding properties of APATMND are discussed with a view towards further development of DNA-binding ligands suitable for gene detection.

Competitive binding of small ligands to nucleobases in AP site-containing DNA duplexes

Kageyama, T., Sato, Y., Nishizawa, S., Teramae, N. — 2008-09-07

By using lumichrome (Lch) as a masking ligand, we successfully control the binding selectivity of 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) when binding to nucleobases in AP site-containing DNA duplexes (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/3'-AGA CGC AGG TCN CGT TGC GTG TG-5', X = AP site; Spacer C3, N = C or T). In solutions buffered to pH 7.0 (I = 0.11 M, at 5°C), ATMND binds to cytosine and thymine with a comparable binding affinity (K_d / nM: C: 7.7, T: 15). By contrast, in the presence of Lch, ATMND shows a clear binding selectivity for cytosine over thymine (K_d/nM: C: 17, T: 204). Such competitive binding events are discussed with a view towards development of ligand-based fluorescence assay for single-nucleotide polymorphisms (SNPs) typing.

Development of label-free molecular beacons based on abasic sitebinding fluorescence molecules

Sato, Y., Nishizawa, S., Teramae, N. — 2008-09-07

Here we report on a class of label-free molecular beacons (MBs) based on a non-covalent interaction with abasic site (AP site)-binding fluorescence molecules. In contrast to conventional MBs that require the chemical labelling with fluorophores and quenchers, our MB simply contains the AP site in the stem moiety, so that a small molecule specifically binds to the AP site. This binding event is accompanied by a significant quenching of its fluorescence, and thus a closed state of the AP site-containing MB (APMB) shows almost no fluorescence. Upon hybridization with a complementary DNA, APMB undergoes a conformational change to take an open state, resulting in an effective fluorescence enhancement due to a release of the molecule from the AP site. These sensing functions of APMB are discussed with a view towards further development of gene detection chemistry based on DNA-binding small molecules.

A surface plasmon resonance sensor based on 3,5-diaminopyrazine with a high selectivity for thymine in AP site-containing DNA duplex

Miura, S., Ono, K., Watanabe, M., Nishizawa, S., Teramae, N. — 2008-09-07

We here report on a surface plasmon resonance (SPR) sensor carrying small organic ligands for the detection of single-nucleotide polymorphisms (SNPs). Two kinds of ligands are prepared, both of which have a hydrogen-bond forming site suitable for nucleobase recognition, and have an active amino group for the immobilization to the sensor chip. While the sensor immobilized flavin does not show any useful responses, the sensor based on 3,5-diaminopyrazine shows a highly selective response to thymine over other nucleobases opposite an abasic site in DNA duplexes (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5', X = AP site, N = target; G, C, A, T). In PBS buffer (pH 6.4, 0.25 M NaCl, at 5°C), the sensor can detect 10 nM of the sample solution, and the SPR signal for thymine is linear in the concentration range from 10 nM to 100 nM. These sensing functions of the present sensor are discussed for the development of SNPs detection chemistry based on DNA-binding small molecules.

Mismatch recognition in PNA double-duplex invasion

Miyajima, Y., Ishizuka, T., Komiyama, M. — 2008-09-07

Double-duplex invasion of pseudo-complementary PNA (pcPNA) to double-stranded DNA is promising for recognition of a specific sequence in double-stranded DNA. In order to apply this process for various purposes such as gene suppression, one base-pair change at the target site in DNA must be strictly distinguished by the pcPNA additives. In this study, mismatch-recognizing activity of double-duplex invasion was investigated under various salt conditions. It has been found that the mismatch-recognition of the invasion is strict enough to distinguish one base-pair alternation, as long as the invasion is achieved in the media of appropriate ionic strength (e.g., [NaCl] = 20 mM).

Crystal structures of DNA duplexes stabilized by bicyclic-C residues

2008-09-07

Chemical modification of nucleic acids is being studied extensively as an approach for the development of nucleic acid-based therapies. We found that a nucleotide carrying 7,8-dihydropyrido[2,3-d]pyrimidin-2-one (bicyclic-C or X), which is a cytosine derivative with a propene attached at the N4 and C5 atoms, increases the stability of DNA duplexes. To establish the conformational effects of X on DNA and to obtain insight into the correlation between the structure and stability of X-containing DNA duplexes, the crystal structures of [d(CGCGAATT-X-GCG)]₂ and [d(CGCGAAT-X-CGCG)]₂ have been determined at 2.9 Å resolutions. In both duplexes, the bicyclic-C bases form pairs with the counter bases through hydrogen bonds, and stabilize the duplex formation in part by stacking interactions between X and the subsequent thymine base of the same strand.

Self-Avoiding molecular Recognition Systems (SAMRS)

Hoshika, S., Chen, F., Leal, N. A., Benner, S. A. — 2008-09-07

Reported here is a "Self-Avoiding Molecular Recognition Systems" (SAMRS), a species of DNA that can bind via simple rules to natural DNA but cannot bind to other members of the same SAMRS species. A system having these properties has been achieved with 2-aminopurine-2'-deoxyriboside (A*), 2'-deoxy-2-thiothymidine (T*), 2'-deoxyinosine (G*) and N4-ethyl-2'-deoxycytidine. These were designed to form more stable base pairs with natural complements than with SAMRS complements, based on the number of hydrogen bonds. Thermal melting studies were performed using duplexes containing SAMRS components. All SAMRS species, A*, T*, G* and C*, formed more stable base pairs with natural complements, T, A, C and G than with SAMRS complements, T*, A*, C* and G* respectively. This property of SAMRS would be useful for avoiding to be produced undesired products derived from intra- and intermolecular interaction between primers in multiplexed polymerase chain reactions.

Finding at-DNA - Kinetic Recognition of Long Adenine-Thymine Stretches by Metal-Ligand Complexes

Nordell, P., Westerlund, F., Reymer, A., Norden, B., Lincoln, P. — 2008-09-07

High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.

Unlocked nucleic acid (UNA) and UNA derivatives: Thermal denaturation studies

Jensen, T. B., Langkjaer, N., Wengel, J. — 2008-09-07

A study on the thermal stability of duplexes formed between unlocked nucleic acid (UNA) modified DNA and RNA oligonucleotides and complementary DNA and RNA is presented. The acyclic UNA monomers are shown to induce a decrease in duplex thermal stability.

Synthesis of 3'- and 6'-functionalised Locked Nucleic Acid (LNA) analogues

Sharma, P. K., Enderlin, G., Kumar, S., Nielsen, P. — 2008-09-07

Two locked nucleic acid (LNA) analogues, i.e. a 3'-C-hydroxymethyl derivative and a 6'(R)-hydroxymethyl derivative of the LNA thymidine monomer, have been synthesized efficiently using convergent strategies. The hydroxymethyl group at the 3'-position or the 6'-position provides a versatile starting point for various modifications of LNA.

Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase

Uchida, Y., Taniguchi, Y., Aoki, E., Togo, M., Sasaki, S. — 2008-09-07

Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-βT and WNA-βC, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-β3AP, having 3-aminopyrazole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-β3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-βC did not show the stabilizing effect.

Strand invasion properties and serum stability of {alpha}-tricyclo-DNA

Scheidegger, S. P., Leumann, C. J. — 2008-09-07

β-tricyclo DNA (tcDNA) is a third generation antisense oligonucleotide developed and studied extensively in our laboratory. We recently became interested in the pairing properties of its -anomeric form and observed parallel duplex formation with natural DNA and RNA. These duplexes were of the same level of thermal stability as their natural counterparts. In addition -tcDNA exhibited poor thermal stability of antiparallel duplexes within its own backbone series which makes it a canditate for double strand invasion applications. Here we report on the strand invasion properties of -tc-DNA and its stability in human serum.

Single-strand binding protein enhances invasion of a PNA strand to double-stranded DNA

Ishizuka, T., Komiyama, M. — 2008-09-07

We show that, in the presence of single-strand binding protein (SSB), one strand of peptide nucleic acid (PNA) efficiently invades double-stranded DNA (dsDNA) in sequence-specific manner, as shown in Figure 1. In the absence of SSB, this kind of strand invasion never occurs. This significant enhancement of invasion can be applied to in vivo applications.

Introduction of linkers into PNA for versatile applications

Aiba, Y., Komiyama, M. — 2008-09-07

PNA (peptide nucleic acid) is widely used as a DNA recognition tool. In this study, we introduced linkers in the middle of PNAs to expand their applicability. By means of Tm measurement and gel-shift assay, it was confirmed that these PNA can recognize double stranded DNA through efficient invasion.

Iso-Thioacetamido nucleic acids (isoTANA): Synthesis and DNA binding studies of Lyxo/Ribo oligonucleotides

Gokhale, S., Kumar, v. A. — 2008-09-07

In the previous study, we developed a method to synthesize 5-atom thioacetamido linked TANA dimer blocks. Incorporation of the dimers in chimeric TANA-phosphodiester linked oligomers and the binding studies towards DNA/RNA preferences were reported. The selectivity towards RNA was found to be significantly high compared to DNA. The lyxo isomer of 3'-amino substitution in thymine was found to have higher 3'-endo conformation in monomer units. In this paper we present studies on synthesis of 3'-lyxo-3'-amino isomer of TANA, its tst and cst dimer units, and incorporation in oligomers. Preliminary results on DNA/RNA binding of these new backbone modified oligomers will be presented.

Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy

2008-09-07

A new approach is described for the insertion of nitroxide spin-labels at specific positions within DNA oligomers. The latter bioconjugaison strategy is based on a click chemistry 1,3-dipolar cycloaddition between a spin-labeling reagent, namely the 4-azido-TEMPO, and alkyne modified uridine-containing oligonucleotides. This highly efficient labeling method was applied for site-specific incorporation of two TEMPO units within a set of double-stranded DNA constructs. Then the determination of the inter-nitroxide distances was achived by using a four-pulses DEER technique that successfully validates the new site-directed spin labeling strategy.

A click chemistry approach towards nucleic acid major groove functionalization

2008-09-07

A synthetic strategy towards new aromatic nucleoside derivatives introducing additional aromatic functionality placed in the major groove of a modified DNA duplex is precented. The functionalities are introduced using Click Chemistry conditions and found to increase the overall duplex stability.

Dynamics of 2-N-tert-Butylaminoxyladenosine Incorporated into Oligodeoxynucleotides

Aso, M., Kurita, M., Mirc, J. W., Kaneko, T., Higuchi, Y., Koga, N., Suemune, H. — 2008-09-07

We synthesized oligodeoxynucleotide (ODN, 3) containing 2-N-tert-butylaminoxyladenosine (1) and studied EPR spectra of 1 and its duplexes with varied sequences. Duplex formation resulted in a broadening of spectrum and a significant decrease in peak-to-peak height and peak height ratio values. The h₊/ho values in EPR spectra well correlated with stability of duplex which indicated ODN containing 1 has the potential to monitor DNA structure.

Nitroxide Spin labeled RNA for long range distance measurements by EPR-PELDOR

Frolow, O., Endeward, B., Schiemann, O., Prisner, T. F., Engels, J. W. — 2008-09-07

Long range distance measurement on RNA allow the determination of RNA folds. Here we report the site specific incorporation of nitroxide spin labels at U,C and A by "on column synthesis". PELDOR (Pulsed Electron Double Resonance) measurements of several RNAs in the range of 2-6nm were successful.

New triarylamine-based far-red DNA stainers with high two-photon absorption properties

2008-09-07

A series of red emitting vinyl-triphenyamines (TP) have been synthesized and evaluated for their two photon absorption (2PA) properties. These compounds are virtually non fluorescent in the free state but exhibit a bright red fluorescence upon binding to doublestranded DNA, both in one- and two-photon absorption. This feature allows one- and two-photon confocal imaging in cells of nuclear DNA with an excellent contrast. Derivatizable analogues for covalent bioconjugation to oligonucleotides are described and variation on the structure is discussed.

Role of Molecular Crowding in perturbing Quadruplex-Watson Crick Duplex Equilibrium.

Kumar, N., Maiti, S. — 2008-09-07

Herein, we address the sensitivity of the competitive equilibria between hoogsteen bonded quadruplex structure and hydrogen bonded Watson-Crick duplex structure. We used osmolytes as molecular crowding agents to mimick intracellular milieu and analysed their effect on Quadruplex-Duplex transition. We used telomeric quadruplex 5'Fluorescein-d[(G₃ TTA)₃ G₃] as a model system and performed extensive Fluorescence Resonance Eenergy Transfer analysis for duplex formation in absence and presence of different concentrations of osmolytes (Glycerol and Ethylene Glycol). Overall the data shows that these molecular crowding agents stabilize quadruplex structure, delays duplex formation and thereby shifts the equilibrium towards quadruplex formation.

Protein hnRNPA1 binds to a critical G-rich element of KRAS and unwinds G-quadruplex structures: implications in transcription

Xodo, L., Paramasivam, M., Membrino, A., Cogoi, S. — 2008-09-07

The promoter of the KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) forming G-quadruplex structures that are recognized by nuclear proteins: PARP-1, Ku70 and hnRNPA1. Here we have studied the interaction between hnRNPA1 (and its derivative UP1) and the G-quadruplexes of KRAS by EMSA, FRET and CD experiments. FRET and CD showed that hnRNPA1/UP1 is able to unfold the G-quadruplexes of KRAS and facilitate the quadruplex to duplex transformation. This finding strengthens our previous hypothesis that the transcription regulation of KRAS is mediated by G-quadruplex structures. Against this background we designed G4-decoy oligonucleotides specific for KRAS that exhibit a strong antiproliferative effect in pancreatic cancer cells.

Structural stability analysis of the intermediates in the folding pathway of human telomeric hybrid-1 G-quadruplex based on fragment molecular orbital method

Yagi, H., Mashimo, T., Sannohe, Y., Sugiyama, H. — 2008-09-07

The human telomeric DNA sequence d[AGGG(TTAGGG)₃] has been found to form different type of G-quadruplex structure based on NMR¹, X-ray crystallography² and circular dichroism (CD). Recently human telomeric hybrid-1 G-quadruplex structure in K⁺ solution has been revealed by CD and NMR^3,4,5. However, folding pathway of G-quadruplex structures is not clear to date. It is important to elucidate the intermediate structure of human telomeric hybrid-1 G-quadruplex for drug discovery in addition to having essential knowledge of telomere. In this study, we designed two types of triplex intermediate model from hybrid-1 NMR structure and evaluated their stabilities with ab initio Fragment Molecular Orbital (FMO) method^6,7,8. The folding pathways of human telomeric hybrid-1 G-quadruplex structure are discussed.

New platinum(II) complexes targeting the loops of the human telomeric G-quadruplex.

Bertrand, H., Bombard, S., Monchaud, D., Teulade-Fichou, M.-P. — 2008-09-07

Two novel series of platinum(II) complexes have been designed and shown to interact with the human telomeric G-quadruplex-DNA via different binding modes: i- terpyridine-platinum (Pt-tpy) complexes covalently interact with quadruplex-DNA via selective platination of adenine residues of the loops, their interaction being driven by the aromatic surface of the ligand; ii- platinum-quinacridine hybrid (Pt-MPQ) interacts with quadruplex-DNA via a dual noncovalent/covalent binding mode, targeting preferentially guanines constitutive of external G-quartets. Therefore, platinum complexes presented herein constitute potential agents for irreversible trapping of G-quadruplex. DNA.

Ligand binding to tetra-end-linked (TGGGGT)4 G-quadruplexes: an electrospray mass spectroscopy study

2008-09-07

The binding properties of a series of known G-quadruplex ligands have been studied by ESI-MS experiments. The tetramolecular (TG₄T)₄ quadruplex and its analogues I and II blocked, respectively, at the 3' or 5'-end by a tetra-end-linker (TEL) unit were chosen as the ligands targets. The stoichiometries of the obtained complexes as well as the ligand affinity and selectivity to the different quadruplexes were determined to deduce the ligand binding site. The TEL derivatives I and II allowed the probing of the grooves contribution to the binding of ligands to G-quadruplexes, demonstrating that the 3' and 5' quartets are not equivalent binding sites for ligand end-stacking.

A short C-rich PNA fragment capable to form novel G-quadruplex-PNA complexes

2008-09-07

In this work we investigated the interaction between the short ac₄a C-rich peptide nucleic acid (PNA) probe and two intramolecular G-quadruplex targets having the same G-tetrad core, but different folding topologies. The T(G₄T)₃G₄T and the recently reported tetra-end-linked-(TG₄T)₄ G-rich oligonucleotides (GROs) were chosen and synthesized for this study. UV, CD, and MS experiments revealed the formation of novel 1:1 G-quadruplex-PNA complexes besides the expected DNA-PNA heteroduplexes.

Human telomere RNA and DNA form an intermolecular G-quadruplex

Xu, Y., Kimura, T., Komiyama, M. — 2008-09-07

For a long time, telomeres have been considered to be transcriptionally silent. Recently, Azzalin et al. demonstrated that telomeres are transcribed into telomeric repeat-containing RNA (TERRA) in mammalian cells. The telomere RNA was found to localize at the telomere DNA. These findings raise a possibility of that telomere RNA may be involved in an intermolecular G-quadruplex with the telomere DNA. In the current studies, we found that human telomere RNA and telomere DNA sequence can form hybrid-type parallel G-quadruplex structure. These results provide valuable information to allow understanding of the roles of human telomeric RNA in chromosome ends regulation and protection.

Orientation of ends of G-quadruplex structure investigated with end-extended oligonucleotides

2008-09-07

The human telomere terminus can adopt the structure of a G-quadruplex. This structure has become an attractive target for anticancer drugs, because it effectively inhibits telomerase activity. In this study, we investigated the orientation of both 5' and 3' ends of the stable G-quadruplex structure. To verify the orientation, we designed end-extended G-quadruplex forming oligonucleotides. We carried out gel electrophoresis and the NMR analysis and found that the ends of the stable G-quadruplex structure are located on opposite faces of each of the quadruplexes.

Molecular docking study of binding of TMPyP4 to a bimolecular human telomeric G-quadruplex

Ishikawa, Y., Fujii, S. — 2008-09-07

We carried out molecular docking simulation of binding of cationic porphyrin TMPyP4 to a bimolecular human telomeric G-quadruplex using DOCK6.1 to examine whether or not to reproduce the loop binding mode of TMPyP4 in the crystal structure. The simulation gave the two highest-ranked docking poses of TMPyP4, and the binding modes were external stacking on the terminal guanine-tetrad and groove binding.

Human Telomeric RNA in G-quadruplex Structure

Xu, Y., Kaminaga, K., Komiyama, M. — 2008-09-07

Very recently, a breaking finding from two groups demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA in mammalian cells. Telomeric RNA, a newly appeared player in telomere biology, may be a key component of telomere machinery. However, structure and function of the telomeric RNA in chromosome ends have not yet been elucidated. In the current studies, we found that the human telomeric RNA sequence can form parallel G-quadruplex structure in the presence of K⁺ or Na⁺ ions. These results provide valuable information to allow understanding of the roles of human telomeric RNA in chromosome ends regulation and protection.

Studies on the influence of inversion of polarity sites on the dG residues glycosidic conformation in quadruplex structures

Esposito, V., Oliviero, G., Pepe, A., Virgilio, A., Galeone, A. — 2008-09-07

Insights into the influence of inversion of polarity sites on the dG residues glycosidic conformation in quadruplexes is presented. The NMR studies concern modified oligodeoxynucleotides based on the quadruplex forming sequence TGGGT.

Structural analysis of r(GGA)4 found in RNA aptamer for bovine prion protein

2008-09-07

RNA aptamers for bovine prion protein (bPrP) were obtained by in vitro selection. It was found that the r(GGA) triplet repeat was frequently present in these aptamers. We already reported that both DNA and RNA containing the GGA repeat form unique quadruplex structures. The unique structures may be utilized for the recognition of bPrP by these aptamers. One of these aptamers contains four continuous repeat of r(GGA), r(GGA)₄. Here, we analyzed the structure of r(GGA)₄ under physiological ionic conditions by NMR. It was revealed on the basis of characteristic NOEs that an r(GGA)₄ strand forms the quadruplex composed of one G:G:G:G tetrad plane and one G(:A):G:G(:A):G hexad plane. Furthermore, two monomers form a dimeric structure in a tail-to-tail manner. The dimeric quadruplex structure of r(GGA)₄ is similar to that of d(GGA)₄, but the difference between two structures was also noticed.

NMR structural study of DNA oligomers containing alkylene crosslinked cyclic 2'-deoxyuridylate dimers

Furuita, K., Murata, S., Jee, J., Ichikawa, S., Matsuda, A., Kojima, C. — 2008-09-07

A 2'-deoxyuridylate dimer cyclized via cross-linkage by ethylene (U^et_pU) or propylene (U^pr_pU) linker was incorporated in DNA oligomer. Fluorescence resonance energy transfer (FRET) experiment showed that they bent at a sharp angle of approximately 90 degree. HMGB1 A-box protein, which selectively binds to bent DNA, binds to the U^et_pU DNA oligomer with high affinity, but not to the U^pr_pU. In order to explain this difference, we have studied the solution structures of the U^et_pU and U^pr_pU DNA oligomers using NMR. Most ¹H signals except for 4', 5' and 5'' were assigned by ¹H-¹H two-dimensional NMR spectra and natural abundance ¹H-¹³C HSQC spectra. Cross-peak patterns of ¹H-¹H NOESY spectra indicate that both oligomers have right-handed B-form DNA like structures and the cyclization in 2'-deoxyuridylates by alkylene crosslinking does not break Watson-Crick base pairs. Chemical shift differences between these two DNA oligomers are localized to the region of 2'-deoxyuridylate dimer and its 3' side. These chemical shift differences and some characteristic NOE crosspeaks suggest the presence of the local structural differences in these regions between the U^et_pU and U^pr_pU DNA oligomers.

NMR assignments and the identification of the secondary structure of the anti-retroviral cytidine deaminase

2008-09-07

APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) is known to have a role in intrinsic cellular immunity against human immunodeficiency virus type1 (HIV-1). The antiretroviral activity of APOBEC3G (APO3G) is associated with the hypermutation of viral DNA through cytidine deamination. APO3G contains two cytidine deaminase domains that are characterised by highly conserved zinc-coordinating motif. It is known that only the C-terminal domain of APO3G (c-APO3G) has the catalytic activity. To shed light on the molecular mechanism of action by which APO3G inactivates HIV-1, we have undertaken the structural and binding studies by NMR. Here, we show the achievement of backbone assignments of c-APO3G and the identification of the secondary structure deduced from chemical shift index (CSI) and NOE data.

Inhibition of human papillomavirus replication by using artificial zinc-finger nucleases

Mino, T., Mori, T., Aoyama, Y., Sera, T. — 2008-09-07

Recently, we have designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined a new approach to inhibition of DNA virus replication by using an AZP-nuclease fusion. In transient replication assays for HPV-18, the genedelivered AZP-nuclease fusion reduced the viral DNA replication rate significantly. Moreover, it was demonstrated by ligation-mediated PCR that viral DNA regions close to the AZP-binding site were cleaved in the cells by the AZP-nuclease. Thus, our results demonstrate that AZP-nucleases have potentials to inhibit replication of any DNA viruses whose replication mechanisms remain unsolved.

Modulation of endogenous VEGF-A expression under hypoxia by using artificial transcription factors

Mori, T., Sasaki, J., Aoyama, Y., Sera, T. — 2008-09-07

The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To modulate the endogenous gene expression artificially, we previously designed a six-finger AZP to recognize a 19-bp DNA in the VEGF-A gene, and fused the AZP with a nuclear localization signal and a repressor domain to generate an artificial transcription factor (ATF). Using the ATF, we demonstrated efficient modulation of the VEGF-A expression. In the present study, we evaluate the ability of the ATF to modulate the gene expression under hypoxic conditions. Enzyme-linked immunosorbent assays (ELISA) for VEGF-A protein in the culture medium revealed that the gene-delivered ATF also repressed the expression of the endogenous VEGF-A gene under hypoxia.

Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins

Koshino-Kimura, Y., Takenaka, K., Domoto, F., Aoyama, Y., Sera, T. — 2008-09-07

Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding. In gel shift assays, we confirmed that the designed AZP has a higher affinity to the replication origin than that of Rep and that the AZP effectively inhibited the Rep binding to its replication origin in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. We will discuss properties of the AZP-transgenic plants against TYLCV infection.

Synthesis of locked S-type and locked N-type uridine monomer units for incorporation in 2'-5' RNA:3'-5'RNA duplexes

Gunjal, A. D., Erande, N., D'Costa, M., Kumar, V. A. — 2008-09-07

To delineate the structural requirements of 2'-5' RNA:3'-5' RNA duplexes, we report the synthesis of N-type locked, S-type locked and 3'-fluoro-2'-phosphoramidite building blocks. The consequent incorporation of these three novel monomers into 2'-5' linked oligomers and their biophysical implications on the stability of the said duplexes will have explicit importance towards development into therapeutic oligomers. The intrinsic stability of 2'-5' phosphodiester linkage as opposed to 3'-5' linked oligomers will be an added advantage

Structural analysis of Musashi-RNA complex on the basis of long-range structural information

2008-09-07

Musashi protein is supposed to be involved in the regulation of differentiation of neural stem cells. Musashi binds to 3' untranslated region of target mRNA and represses the translation of mRNA. Musashi has two tandem RNA-binding domains (RBDs), RBD1 and RBD2. Both RBDs cooperatively bind to the target mRNA. Here, we determined the structure of RBD1-RBD2 in complex with target RNA. First, the structures of two RBDs in the complex were determined on the basis of short-range distance restrains derived from NOEs. However, the relative position of two RBDs was not determined due to the lack of long-range distance restraints across two RBDs. In order to overcome the situation, we introduced the paramagnetic center into Musashi by attaching MTSL carrying the NO radical. The long-range distance restraints (ca. 20–40 Å) between two RBDs were derived from paramagnetic relaxation enhancement (PRE) caused by the paramagnetic center. The relative position of two RBDs was successfully determined on the basis of these longrange distance restraints. The change in the relative position of two RBDs on binding to the target RNA was also detected by PRE. The determined structure of RBD1-RBD2 in the complex has suggested how Musashi recognizes its target mRNA.

Construction of a stable functional ribonucleopeptide complex by the covalent linking method

Fukuda, M., Nakano, S., Tainaka, K., Fujieda, N., Morii, T. — 2008-09-07

We describe here a novel strategy to create a stable functional ribonucleopeptide (RNP) complex by the covalent linking method. Adenosine-5'-triphosphate (ATP)-binding RNP receptors were selected from the RNP library by in vitro selection. The RNA subunit of RNP is utilized to construct a ligand-binding cavity, while the peptide subunit can be functionalized independently. By introducing a fluorophore at the N-terminus of the Rev peptide subunit, the ATP-binding RNP receptor is successfully converted to a noncovalent complex of ATP-responsive fluorescent RNP sensor. Such a noncovalent RNP sensor could be covalently linked by the tethering the RNA to the fluorophorelabeled peptide subunit to form a stable RNP sensor without losing the original function.

The specific interaction between metal cation and mismatch base pair in duplex RNA

Kozasa, T., Miyakawa, Y., Ono, A., Torigoe, H. — 2008-09-07

We have already found that mercury (II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4°C. We have also found that silver (I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4°C. In the present study, to examine whether the specific interaction between metal cation and mismatch base pair can be also formed in duplex RNA, we investigated the effect of the metal cation on the thermal stability of homoduplex and heteroduplex RNA. Addition of mercury (II) cation increased the melting temperature of heteroduplex RNA containing U:U mismatch base pair by about 6°C. The thermal stability of homoduplex RNA containing U:A or A:U perfectly matched base pair and heteroduplex RNA containing A:A mismatch base pair was not significantly changed by the addition of mercury (II) cation. On the other hand, addition of silver (I) cation increased the melting temperature of heteroduplex RNA containing C:C mismatch base pair by about 4°C. The thermal stability of homoduplex RNA containing C:G or G:C perfectly matched base pair and heteroduplex RNA containing G:G mismatch base pair was not significantly changed by the addition of silver (I) cation. We conclude that the specific interaction between metal cation and mismatch base pair can be formed in duplex RNA as in the case of duplex DNA.

Selective recognition of a tetra-amino-acid motif containing phosphorylated tyrosine residue by ribonucleopeptide

Nakano, S., Hasegawa, T., Fukuda, M., Fujieda, N., Tainaka, K., Morii, T. — 2008-09-07

We describe here a ribonucleopeptide (RNP) receptor targeting a tetra-amino-acid motif containing phosphotyrosine, GpYSR. GpYSR-binding RNP receptors were obtained from an RNA-based RNP library by in vitro selection. These receptors have a higher affinity than those of previously obtained pY-binding RNP receptors. One of these RNP receptors exhibited unique specificity to the target GpYSR peptide over other tetra-amino-acid peptides derived from the tyrosine-phosphorylation sites of native proteins. The GpYSR-binding RNP receptor discriminated not only the phosphorylated tyrosine residue, but also its surrounding three amino acid residues. Thus, RNP receptors could target a defined pY-containing amino-acid sequence by expanding the recognition surface within the ligand-binding pocket of RNP.

Development of ribonucleopeptide-based fluorescent sensors for biologically active amines based on the stepwise molding strategy

Tainaka, K., Hasegawa, T., Fukuda, M., Nakano, S., Fujieda, N., Morii, T. — 2008-09-07

General strategy for the development of fluorescent biosensors as a tracer of ‘key’ molecule in the cellular system would provide important breakthroughs for ubiquitous applications in the field of diagnosis and pharmacology in addition to our understanding of cellular events. The sophisticated design of fluorescent biosensors based on the organic synthesis is one of the promising approaches, but this type of biosensors frequently fail to maintain their performance in the cellular environment despite of laborious protocols. Another procedure for simultaneous preparation of a wide variety of fluorescent biosensors for the optical monitoring of a target molecule represents an especially attractive alternative. In our continuous efforts, we have recently developed a conceptually new strategy for coincidental production of fluorescent biosensors with diverse functions based on a framework of ribonucleopeptide (RNP). RNP-based fluorescent sensors were fabricated with a combination of in vitro selection method and a successive modification of the peptide of RNP with a fluorophore. Each RNP composed of a ligand-binding RNA subunit and a fluorophore-tagged peptide motif facilitated the fluorometric detection of biologically active amines with a unique binding affinity and an inherent fluorescent signal.

Significance of the N-terminal Histidine-rich Region for the Function of the Human Toll-like Receptor 3 Ectodomain

2008-09-07

Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments such as endosomes and signals in response to virus-derived double-stranded RNA (dsRNA). TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To clarify the pH-dependent binding mechanism of TLR3 at the structural level, we focused on 3 highly conserved histidine residues clustered at the N-terminal region of the TLR3 ectodomain (ECD): H39, H60 and H108. Mutagenesis of these residues showed that H39, H60, and H108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to the recombinant TLR3 ECD depended strongly on pH and dsRNA length, and was reduced by mutations of H39, H60, and H108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA that incorporates this second binding site.

Isolation of RNA aptamers specific for the 3' X tail of HCV

2008-09-07

The 3' end of the HCV genome, designated as the 3' X tail, comprises an almost invariant 98-nucleotide sequence containing three highly conserved stem-loop structures (3' SL1, 3' SL2, and 3' SL3). Since these sequences are all critical for the initiation of negative-strand synthesis and essential for viral replication, they are attractive targets for novel anti-HCV drugs. To obtain effective RNA aptamers specific for the 3' X tail, and with the aim of developing novel inhibitors of HCV replication, we performed in vitro selection of aptamers with specificity for the 3' X tail. In vitro selection, namely SELEX (systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating nucleic acid sequences from a randomized oligonucleotide pool that have a high affinity for a target molecule. After four selection cycles, a pool of the 3' X tail-specific RNA aptamers were obtained. This RNA pool included 39 clones that could be divided into three main classes (cSL1, cSL2, and cSL3) which harbor complementary sequences to the apical loops of 3' SL1, 3' SL2, and 3' SL3, respectively. Biochemical analyses are in progress to evaluate whether these RNA aptamers have the potential to block HCV replication.

Subsites for substrate recognition by bacterial ribonuclease P

Fujimoto, A., Suwa, S., Nagai, Y., Kikuchi, Y., Tanaka, T. — 2008-09-07

We have prepared series of shape variant RNAs of a tRNA precursor and analyzed the substrate shape recognition by bacterial ribonuclease P ribozyme and holoenzyme. The results showed the evidence for the presence of subsites for the recognition of the trna shape. We will discuss a new model for substrate recognition and the role of the protein component.

Analysis of the interaction between selected RNA-binding peptides and a target RNA containing a bulge and a GNRA-type tetraloop

Horiya, S., Koh, C.-S., Matsufuji, S., Harada, K. — 2008-09-07

We have characterized the interaction between selected novel RNA-binding peptides and their target RNA. The RNA is comprised of two elements, a GCAA tetraloop, a member of the thermodynamically stable GNRA-type (where N is A or G, U, C; R is G or A) tetraloops, and a tri-purine bulge found in the frameshift stimutating structure on the human immunodeficiency virus type 1 (HIV-1) gag-pol mRNA. Peptides that bind specifically to the target RNA were selected from a combinatorial library based on arginine-rich motif (ARM) by a bacterial reporter system. We performed mutational studies using the reporter system and gel shift assays and found that the binding affinity and specificity of the RNA were mainly dependent on the GNRA-type tetraloop, and a modest contribution was also attributed to the bulge structure. Our finding reveals a novel mode of interaction by an RNA-peptide complex and expands our knowledge on the diversity of molecular recognition.

Fluorescent Ligand as a molecular probe for the RNA structure

Umemoto, S., Zhang, J., Dohno, C., Nakatani, K. — 2008-09-07

In this report, we show the recognition of RNA secondary structure by exploiting a fluorescent 2,7-disubstituted 9H-xanthen-9-one derivative (2). The xanthone derivertive (2) especially bound to RNA and became non-fluorescent. Upon mixing with RRE model RNA, U-bulge RNA, or hairpin RNA, the fluorescence intensity of 2 was decreased in different intensity. The distinct fluorescent increase of 2 to each RNA seems to a good probe for The RNA structures.

Identification of RNA Binding Specificity for the TET-family Proteins

Takahama, K., Kino, K., Arai, S., Kurokawa, R., Oyoshi, T. — 2008-09-07

The TET-family proteins (TAF15, EWS and TLS) are the RNA binding proteins involved in multiple levels of cellular functions. The RNA binding domain of those proteins is known as the important region for cellular functions. But little is known about the RNA binding specificity of TET-family proteins. In order to investigate the RNA binding properties of the TET-family proteins, we performed electrophoretic mobility shift assay using recombinant Flag-tagged TLS and guanine-rich and RNAs. It was found that TLS binds to human telomeric RNA in the presence of KCl, but not in the presence of LiCl.

Isoenergetic microarray mapping reveals differences in structure between tRNAiMet and tRNAmMet from Lupinus luteus

Kierzek, E., Barciszewska, M. Z., Barciszewski, J. — 2008-09-07

Isoenergetic microarray mapping is shown to be an ideal method for probing subtle structural differences between initiator tRNA_i^Met and elongator tRNA_m^Met from Lupinus luteus. The differences in structure of both tRNAs cause significant dissimilarities in binding to microarrays probes.

Interactions between antitumor drugs and vault RNA

2008-09-07

It is supposed that ribonucleoprotein particle vault is involved in detoxification processes and thus is related to multidrug resistance. The vault is composed of three proteins and three vault RNAs, hvg-1, -2 and -3. The direct interactions between vault components and drugs were not reported. Recently, we revealed the interactions between vault RNAs and mitoxantrone. Here, we examined the interactions between hvg-2 and six antitumor drugs by a chemical shift perturbation method of NMR. It was found that in addition to mitoxantrone, hvg-2 can interact with two drugs basically in the same way using the same site. The difference in the affinity was also noticed among three drugs. Hvg-2 did not bind to the other three drugs. It is suggested that the common or closely related chemical structure of the positive three drugs is recognized by vault RNA.

Isoenergetic microarray mapping - the advantages of this method in studying the structure of Saccharomyces cerevisiae tRNAPhe

Jenek, M., Kierzek, E. — 2008-09-07

Isoenergetic microarrays were applied to study the structure of processed S. cerevisiae tRNA^Phe and its unmodified transcript. Results of hybridization experiments demonstrate significant differences in binding of both RNAs. The microarray mapping approach provides similar structural information as traditional chemical or enzymatic mapping, but is superior in several ways.

Crystal structures of RNA 3'-terminal phosphate cyclase and its complexes with Mg2++ATP, ATP or Mn2+

2008-09-07

RNA 3'-terminal phosphate cyclase (Rtc) is an enzyme related to RNA splicing, in which the 3'-terminal hydroxyl group of a truncated RNA is converted to the 2',3'-cyclic phosphate that is required prior to RNA ligation. This reaction may occur in the following two steps: (i) Rtc + ATP -> Rtc-AMP + Ppi and (ii) RNA-N3' + Rtc-AMP -> RNA-N>p + Rtc + AMP. In order to establish the reaction mechanism, Rtc of Sulfolobus tokodaii, overexpressed in E. coli, was crystallized in the following states, Rtc, Rtc-AMP, Rtc:AMP, Rtc:ATP and Rtc:Mn, and their crystal structures have been determined at 2.25, 2.25, 2.9, 2.4 and 3.2 Å resolutions, respectively. Based on these structures, a possible reaction mechanism has been proposed.

Lipophilic DNA-conjugates: DNA controlled assembly of liposomes

Jakobsen, U., Vogel, S. — 2008-09-07

DNA detection systems based on encoded solid particles have been reported but require often tedious and not generally applicable surface chemistry. In the present study a system comprised of a lipid-modified DNA probe sequence and unmodified DNA target sequences is used to non-covalently assemble liposomes. The process results in large liposome aggregates with dramatically different optical properties compared to individual liposomes in solution. The presented method enables fast and easy detection of target polynucleotides. Furthermore, the system displays remarkably sharp thermal transitions which enable detection of single nucleotide polymorphisms with greatly enhanced resolution compared to flourescence based methods.