Nucleic Acids Symposium Series - current issue

Development of an N-unprotected phosphoramidite method for the chemical synthesis of aminoacylated RNAs

Ohkubo, A., Noma, Y., Taguchi, H., Seio, K., Sekine, M. — 2007-11-20

In the previous study, we developed a method for O-selective phosphorylation, i.e., "the activated phosphite method" for DNA synthesis without base protection. However, the O-selectivity was low in RNA synthesis when the activated phosphite method was used. In this paper, we developed a new method for the O-selective phosphorylation available for the chemical synthesis of aminoacylated RNAs on polymer supports. As the result, it was found that the selectivity of the phosphorylation in RNA synthesis without base protection increased to more than 99% by treatment of the undesired N-P(III) bonds with 1-hydroxyl-6-nitorobenzotriazole, independent of the kind of protecting groups at the 2' position. Moreover, we succeeded in synthesizing a 21 mer oligoRNA without base protection.

Chemical synthesis of a very long RNA oligomer, a 110mer precursor-miRNA candidate, with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group

2007-11-20

A long RNA oligomer, a 110mer with the sequence of a precursor-miRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We confirmed the identity of the synthetic 110mer by MALDI-TOF mass spectrometry, as well as HPLC, electrophoretic methods, RNase-digestion experiments, and its in vitro gene-silencing activity. The chemical synthesis of RNA oligomers of more than 100 nucleotides, which has until now been extremely difficult, can be practically realized by the CEM method.

Development of novel thioguanosine analogs with the ability to specifically modify cytidine

Onizuka, K., Taniguchi, Y., Sasaki, S. — 2007-11-20

Site-specific modification of nucleic acid is of great significance in the machinery of gene expression. Specific modification of nucleic acids by an oligonucleotide incorporating a S-vinyl thioguanosine analog, has great potential as a useful tool. The specific transfer of the vinyl derivative to the amino group of dC at the target site of the complementary ODN has been demonstrated. This is an innovative method for the efficient and selective modification of the target cytidine in DNA

Synthesis, Duplex-forming Ability and Fluorescent Property of OligoDNA Bearing Silylated Fluorophore

Shinozuka, K., Sekiguchi, T., Ebara, Y., Moriguchi, T. — 2007-11-20

Novel pyrene derivative conjugated with dimethylsilyl moiety was prepared from bromopyrene. The compound was successfully converted to the corresponding phosphoramidite derivative and incorporated into oligoDNA. In uv-melting study, the resulted fluorescent oligoDNA can discriminate one-base mismatched complement only when the mismatched base is positioned around the middle of the duplex-forming region. On the other hand, the oligoDNA gives marked fluorescence upon binding with the full-matched complement. The intensity of the fluorescent signal of the oligoDNA in a duplex changes depending on the position of the mismatched base of the complement.

Development of an unnatural base pair for efficient PCR amplification

Hirao, I., Mitsui, T., Kimoto, M., Yokoyama, S. — 2007-11-20

An unnatural base pair system could expand the genetic alphabet, enabling the site-specific incorporation of extra, functional components into nucleic acids and proteins. We developed an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (denoted by Ds) and 2-nitropyrrole (denoted by Pn), which specifically and efficiently functions in DNA amplification by PCR. After 20 cycles of PCR, the mutation rate of the Ds-Pn pair in an amplified DNA fragment was approximately 1%. The Ds-Pn pair system in combination with other unnatural base pairs could be useful for DNA/RNA-based biotechnology.

Construction of novel dye aggregates based on "comb-type" sequence

Kashida, H., Fujii, T., Asanuma, H. — 2007-11-20

Novel dye aggregates ("comb-type" aggregates) was prepared by hybridizing modified oligodeoxyribo-nucleotides (ODNs), in which dyes were introduced consecutively. From NMR structural analysis, dye molecules were intercalated between base pairs and stacked in anti-parallel manner. When ODNs containing three Methyl Red moieties were hybridized, strong exciton coupling was observed. In addition, thermal stability of duplex was substantially enhanced due to the intermolecular stacking.

The Genetic Code Revisited Four Decades after Francis Crick

Soll, D. — 2007-11-20

The various strategies of codon decoding in organisms of the three domains of Life: evolutionary implications

Grosjean, H., Marck, C., de Crecy-Lagard, V. — 2007-11-20

Over a thousand of cytoplasmic, non organellar tRNA genes were extracted from the whole-genomes of more than 100 organisms spanning the Bacteria, Eukarya and Archaea (tRNomics). Also, whenever possible, the genes coding for modification enzymes acting on tRNA, particularly those involved in modification of nucleotides in the anticodon loop, were identified (Modomics). Combining these two data sets, we were able to reveal three main decoding strategies used by individual contemporary organisms to read the 62 (61+1 initiator) sense codons of mRNA. Based on the known phylogenetic relationships of the different organisms analyzed, this work allows to predict which RNA modification enzymes are essential for an accurate and efficient translation process, as well as to shed light on when these complex and diverse tRNA maturation processes probably emerged during cellular evolution.

Property editing of peptide nucleic acids (PNA): gem-dimethyl, cyanuryl and 8-aminoadenine PNAs

Ganesh, K. N, Gourishankar, A., Vysabhattar, R., Bokil, P. — 2007-11-20

We herein describe the introduction of gem-dimethyl substitution into the aminoethylglycyl backbone of PNA to impart steric constraint and pre-organise PNA for selective recognition of nucleic acids. Introduction of cyanuric acid and 8-aminoadenine as pyrimidine and purine analogs that can form base pairing from either face is also described to overcome the rotameric problems in PNA sidechain orientations and thereby enhance the statistical probability for base pairing. The UV-thermal melting studies of the derived triplexes with complementary DNA provide support for this rationale.

A new concept in double duplex DNA invasion by chiral PNAs which simultaneously depress PNA-PNA and improve PNA-DNA duplex stability.

Sforza, S., Tedeschi, T., Corradini, R., Marchelli, R. — 2007-11-20

By using suitably designed chiral, lysine-based PNAs (chPNAs), the stability of complementary chPNAchPNA duplexes can be depressed, maintaining or improving at the same time the stability of the corresponding PNA-DNA duplexes. This approach could be used in order to develop chPNAs able to perform double duplex DNA invasion.

Synthesis of Peptide Ribonucleic Acid (PRNA)-DNA Chimera and Interaction with DNA and RNA

Wada, T., Maeda, Y., Sawa, N., Sato, H., Chon, H., Kanaya, S., Inoue, Y. — 2007-11-20

Recently, we have demonstrated that effective control of the recognition behavior of peptide ribonucleic acid (PRNA) with complementary DNA is possible through the anti-to-synorientational change of pyrimidine nucleobase induced by borate ester formation. In this study, DNA-PRNA chimera was prepared by the solidphase synthesis. In the DNA-PRNA chimeras, both PRNA and DNA domains work as recognition sites for the complementary DNA/RNAs to form stable complex, while DNA-RNA hybrids formed in the DNA domains of DNA-PRNA chimera should be substrates to the hydrolysis by RNase H and PRNA moieties work as recognition control/switching devices and as inhibitor for the hydrolysis by exonucleases. Interaction of the DNA-PRNA chimera with DNA and RNA has been discussed.

Design of dual-labeled oligonucleotide probes for SNPs genotyping

Saito, Y., Bag, S. S., Kodate, S., Suzuka, I. — 2007-11-20

Our effort in designing base-discriminating fluorescence nucleosides (BDF), leads us to develop dual-labeled oligonucleotide probe in which the BDF nucleoside, ^PyU/^2-AntU act as the donor separated by a defined base pair distance from the acceptor, fluorescein, attached to 5'-end of the probe. Thus, a longer wavelength emission from acceptor might allow the probe to be used for SNP typing in chip based detection technology or in cell.

Recognition of triplex forming oligodeoxynucleotides incorporating abasic sites by 5-arylcytosine residues in duplex DNAs

Mizuta, M., Banba, J.-i., Kanamori, T., Ohkubo, A., Sekine, M., Seio, K. — 2007-11-20

In this paper, we reported our attempt to use a 5arylcytosine (dC^ar) and the abasic site () as an artificial base pair for DNA triplex. The idea was confirmed by the molecular modeling studied in which the aromatic group of ^ph which protrudes in the major groove was buried into the cleft formed by the residue in the TFO. We synthesized three kinds of dC^ar and the oligonucleotides incorporating them. Our UV-melting experiments revealed that the DNA triplex containing the dC^ph was more stable than that containing dC pair. Moreover, the dC^ph pair was more stable than any other dC^phY pairs such as dC^phG, dC^phC, dC^phT and dC^phA. These results indicated the possibility that the appropriate pair of dC^Ar and could be the new sequence code of DNA triplex. We also carried out the Tm analyses of other TFOs incorporating dC^Ar and , and clarified the stability of these triplexes.

DNA methylation analysis using metal complex formation

Okamoto, A., Tanaka, K., Tainaka, K., Umemoto, T., Nomura, A. — 2007-11-20

Analysis of the cytosine methylation status of a gene is very important for understanding the expression mechanism of genetic information. However, to distinguish 5-methylcytosine (M) from C, i.e., to detect the existence of only one methyl group in a long DNA strand, is not easy. A rapid and selective chemical reaction capable of distinguishing between M and C would become a useful method for efficiently analyzing the status of cytosine methylation at a specific site in a gene. We herein report M-selective oxidation. M was oxidized efficiently by exposure to a reaction mixture containing an osmium complex, making possible a clear distinction from very weak oxidation of C. We readily obtained information on the methylation status at a specific site by means of M-selective oxidation using functional bipyridine ligands.

Novel Applications of Locked Nucleic Acids

Veedu, R. N., Vester, B., Wengel, J. — 2007-11-20

Locked Nucleic Acid (LNA) nucleoside triphosphates were prepared and their substrate properties for different polymerases during primer extension and PCR experiments investigated. Phusion^TM High Fidelity DNA polymerase and 9^°Nm^TM DNA polymerase readily accept LNA nucleoside 5'-triphosphates as substrates in primer extension assays. However, in PCR assays, However, in PCR assays, DNA 9oN_m^TM polymerase proved to be the best for amplification employing the LNA-A nucleotide.

Self-Organization of Polyaromatic Compounds within DNA

Malinovskii, V. L., Bittermann, H., Haner, R. — 2007-11-20

The structural organization of oligopyrene sections embedded within a DNA framework is described. Absorbance, fluorescence and circular dichroism spectroscopy provide insight into the molecular interactions of the pyrene units.

The photoreactivity of the caged precursors of oligonucleotides containing C4'-oxidized abasic site and reaction of the generated lesion

Usui, K., Aso, M., Fukuda, M., Toguchi, Y., Suemune, H. — 2007-11-20

The C4’-oxidized abasic site (1) is one of the oxidatively damaged DNA lesions by antitumor bleomycins (BLMs) and the acyclic keto aldehyde incorporated in DNA might react with DNA interacting biomolecules. We synthesized oligodeoxynuceotides 3-caged-1 and 3-caged-2 as caged precursors of 3, an ODN containing 1, to study the reactivities of 3 with amine. Irradiation of double stranded 3-caged-1 led to efficient formation of 3 compared to its single stranded form. Similarly, 3-caged-2 and its duplex afforded 3. With 3-caged-2, yields of 3 were lower compared to 3-caged-1. Formation of the duplex slowed decaging rate down but the yield of 3 was higher than that of single stranded 3-cage-2. Reaction of the obtained 3 with amine resulted in the formation of lactam 2b in good yield.

Synthesis and biological properties of pyrrole-imidazole polyamide conjugates

2007-11-20

We have developed a series of conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. High resolution polyacrylamide gel electrophoresis revealed that these conjugates alkylated DNA at predetermined sequences. Then, we demonstrated that conjugates 1 and 2 have DNA alkylation activities at double stranded human telomere sequence and potent cytotoxicities in cancer cell lines. In addition, we showed that conjugate 3 alkylates DNA with ten-base-pair recognition sequence in the presence of partner polyamide 4, which suggested alkylation through 3-4 heterodimer formation.

Construction of supramolecular assemblies and self-organized structures using oligo-DNAs

Ohya, Y., Nishi, T., Nohori, T., Jo, S., Ohta, K., Jozuka, K., Ouchi, T. — 2007-11-20

To obtain fundamental theories for construction of self-organized structures using oligonucleotides, we constructed multiple aggregates with half-sliding oligo-DNAs. We used several types of half-sliding oligo-DNAs with different stability and number of residues. The formation of multiple aggregates by the oligo-DNAs was confirmed by size exclusion chromatography (SEC). The shape of the aggregates was investigated by atomic force microscope (AFM), and linear structures of multiple aggregates of half-sliding oligo-DNAs were observed.

The impact of 8-aryl-and 8-heteroaryl-2'-deoxyguanosine derivatives on G-quadruplex formation

Gubala, V., Rivera-Sanchez, M. d. C., Hobley, G., Rivera, J. M. — 2007-11-20

Guanine and G-rich oligonucleotides are known to self-assemble in the presence of a variety of cations to form higher ordered structures known as Gquadruplexes. We have synthesized a library of 8-aryl/heteroaryl-2'-deoxyguanosine derivatives (8ArGs) that are also able to self-assemble into quadruplex structures. We demonstrate that the properties of such quadruplexes can be modulated by the nature of the groups attached to the guanine base. These supramolecules are potentially useful in the development of self-assembled nanodevices.

Aminoacyl-tRNA surveillance by EF-Tu in mammalian mitochondria.

Nagao, A., Suzuki, T., Suzuki, T. — 2007-11-20

Aminoacyl-tRNA synthetases specifically recognize their cognate tRNAs and ensure the accuracy of translation. However, in mammalian mitochondria, seryl-tRNA synthetase (mt SerRS) significantly misacylates tRNA^Gln, indicating the presence of another mechanism to be required to maintain the fidelity of mitochondrial protein synthesis. We have revealed that mammalian mitochondrial elongation factor Tu (mt EF-Tu) tends to interact with seryl-tRNA^Gln with lower affinity than glutaminyl-tRNA^Gln and seryl-tRNA^Ser. This result proposes that mt EF-Tu has a critical role to maintain the translational fidelity by surveillance of aminoacyl-tRNAs for quality control.

Molecular mechanism of trans-translation

Kurita, D., Konno, T., Takada, K., Muto, A., Himeno, H. — 2007-11-20

tmRNA has a dual function as a tRNA and an mRNA to relieve the stalled ribosome. During this process, tmRNA enters the ribosomal A-site without a codonanticodon interaction, but with a protein factor SmpB. Here, we established in vitro trans-translation system, which is able to evaluate peptidyl transfer to alanyltmRNA and translation of the resume codon on tmRNA. Using this system, the effects of mutations on tmRNA or SmpB are assessed by measuring the incorporations of labeled amino acid into polypeptide. Interaction of SmpB with ribosome and tmRNA was studied by directed hydroxyl radical probing. Our results revealed that SmpB constitutes an important element during trans-translation. We propose a new model of transtranslation.

Kinetic analysis of the effects of translation enhancers in translation initiation

Takahashi, S., Furusawa, H., Shimizu, Y., Ueda, T., Okahata, Y. — 2007-11-20

Translation initiation is the most important step within a series of protein biosynthesis processes because the incorporation of ribosomes to a mRNA mainly determines efficiencies of translation. In bacteria, translation enhancers located on the 5’ upstream of the Shine-Dargalno (SD) sequence on mRNAs are known to accelerate the efficiency of protein biosynthesis. To investigate the role of translation enhancers in translation initiation, we analyzed binding kinetics of a 30S ribosomal subunit to a mRNA immobilized on a 27 MHz quartz-crystal microbalance (QCM). The association constant (Ka) was rather low for the mRNA including a translation enhancer sequence compared with that for the mRNA without translation enhancers. These kinetic parameters suggest that translation enhancers destabilize the ribosome-mRNA complex on an SD sequence to move on the next step of decoding its mRNA.

Chemistry of Bisulfite Genomic Sequencing; Advances and Issues

Hayatsu, H., Negishi, K., Shiraishi, M., Tsuji, K., Moriyama, K. — 2007-11-20

Methylation at position 5 of cytosine in DNA plays a major role in epigenetic gene control. The methylation analysis can be performed by bisulfite genomic sequencing. Conventional procedures in this analysis include a treatment of single stranded DNA with 3–5 M sodium bisulfite at pH 5 and at 50–55° for 4–20 hr. This will convert cytosine into uracil, while 5-methylcytosine resists this deamination. Amplification by PCR of the bisulfite-treated DNA followed by sequencing reveals the positions of 5-methylcytosine in the gene. We reported recently that the whole procedure can be speeded up by use of a highly concentrated bisulfite solution, 10 M ammonium bisulfite. We also reported that urea, which has been often added to the reaction mixture with the purpose of facilitating the reaction, may not work as anticipated. This time, we would like to address the need for further investigating the chemistry of the bisulfite modification of DNA. Particularly important is to study side reactions that may occur due to the exhaustive bisulfite treatment required for achieving complete deamination of all the cytosine residues in a given sample of DNA.

Nucleotide incorporation against 7,8-dihydro-8-oxoguanine is influenced by neighboring base sequences in TLS DNA polymerase reaction

2007-11-20

7,8-Dihydro-8-oxoguanine (8-oxoG) is a well-known oxidative lesion in DNA and is related to carcinogenesis and ageing processes. Misincorporation of dATP opposite to 8-oxoG leads to G -> T transversion mutations. DNA sequence has been proved as an important factor influencing the replication and enzymatic repair of various types of damages. To explore the influence of sequence effect on the properties of translesion synthesis (TLS) polymerase bypass of 8-oxoG, oligonucleotides with an 8-oxoG in different sequence contexts were used. We conclude that the 5'-nearest base next to 8-oxoG has significant effects in the G -> T mutation by hpol.

Base excision repair system suppresses mutagenesis caused by 8-hydroxy-dGTP in Escherichia coli

Suzuki, T., Yamamoto, K., Harashima, H., Kamiya, H. — 2007-11-20

To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) was introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei), and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 1.8-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.

Functional reactivity of oxanine: its biological meanings and biotechnological applications

Pack, S. P., Doi, A., Kamisetty, N. K., Nonogawa, M., Kodaki, T., Makino, K. — 2007-11-20

Oxanine (Oxa), generated as one of the major products from guanine by nitrosative oxidation, has been focused as a mutagenic lesion. Here, Oxa was explored in terms of its unique property to react with – NH2 or –SH group since Oxa possesses O-acylisourea conformation in the base-ring structure. In particular, Oxa has been characterized in terms of its formation and mechanism of DNA-protein cross-link (DPC). In addition, Oxa was testified as a new carboxy-linker for activation-free covalent bonding with NH2-molecules, which can be usefully employed for the design of biotechnological or nano/biotechnological systems.

Polymerisation of a DNA strand using oligo-DNA template with modified bases, sugars and phosphates

Nagashima, J.-i., Minezaki, S., Obika, S., Imanishi, T., Kuwahara, M., Sawai, H. — 2007-11-20

We have attempted to synthesise a complimentary strand using oligo-DNA with modified bases, sugars and phosphates as a template strand by polymerase reaction. Analogues bearing C5-substituted uracil, those with amide linkage [–CH2C=ONH–] in place of phospho-diester linkage and those bearing 2'-O, 4'-C-bridged sugar were used. Primer extension reactions were carried out to synthesise complimentary DNA strands. The reactions depended on the thermostable DNA polymerase used, the type of modification or the number of the modified position on the template strand.

Amplification of 4'-thioDNA and its utility as a shRNA expression system

Inoue, N., Shionoya, A., Ogawa, N., Matsuda, A. — 2007-11-20

In this presentation, we discuss PCR amplification of 4’-thioDNA using 2’-deoxy-4’-thionucleoside triphosphate (sdNTPs). The amplified 4’-thioDNA acted as templates for not only in vitro transcription by T7 RNA polymerase, but also transcription in cells by RNA polymerase III. Accordingly, we succeeded to inhibit gene expression in cells by transfection of 4’-thioDNA that expresses a short-hairpin RNA (shRNA).

Chemical biology that controls DNA structure and function

Sugiyama, H. — 2007-11-20

All genetic information, which is necessary for life, is written in 3 billion base pairs of DNA. Sequencedependent local DNA conformations are also thought to play an important role in gene expression. Many diseases, including cancer, and hereditary and viral diseases, can now be understood at the DNA sequence and structure level. Therefore, direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. In this presentation I focus on our long-term efforts in controlling of structure and function of DNA by chemical biological approach.

Making Oligonucleotide Conjugates and Breaking Oligonucleotides

Milton, S., Murtola, M., Sandbrink, J., Yeheskiely, E., Stromberg, R. — 2007-11-20

ABSTRACT Presented is our most recent work on synthesis and properties of oligonucleotide analogues and conjugates These include 2’-carbamoylmethyl derivatives, peptideoligonucleotide conjugates and conjugates made to combine Watson-Crick and non-Watson-Crick interactions as well as conjugates with RNA-cleaving groups. Recent results on cleavage of RNA by artificial nucleases, is also presented.

Transition State Analogue Inhibitors of N-Ribosyltransferases: New Drugs by Targeting Nucleoside Processing Enzymes.

Evans, G. B., Furneaux, R. H., Kelly, P. M., Schramm, V. L., Tyler, P. C. — 2007-11-20

The characterization of the transition state structure of a number of N-ribosyltransferases has enabled the design and synthesis of some extremely powerful inhibitors of these enzymes. We have three generations of inhibitors for some nucleoside processing enzymes which are therapeutic targets, and the potency of these compounds confers special advantages in their development as new drugs against cancer, autoimmune diseases, microbial infections and malaria.

Metal ion promoted cleavage of RNA phosphodiester bonds: from Zn(II) aqua ion to artificial ribonucleases

Wang, Q., Niittymaki, T., Virta, P., Ketomaki, K., Mikkola, S., Lonnberg, H. — 2007-11-20

The potential of Zn²⁺ azacrown chelates as constituents of artificial ribonucleases is discussed.

Multivalent Catalysts for the Cleavage of Nucleic Acids and their Models

Manea, F., Pasquato, L., Prins, L. J., Scrimin, P. — 2007-11-20

Multivalent systems, i.e. systems presenting a discrete collection of several functional groups, are well recognized for their ability to increase binding constants. Less explored is their use as catalysts. We present a number of multivalent nanostructures based on self-assembled gold nanoparticles and dendrimers, which are particularly efficient in the cleavage of nucleic acids and their models.

Conformationally-2',4'-Locked Aza-ENA and Carbocyclic ribo-Thymidine

Chattopadhyaya, J. — 2007-11-20

AONs containing aza-ENA (1), 5-membered (2) and 6-membered (3) carbocyclic analogs of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) are both nuclease resistant and capable of eliciting RNase H response, very similar to that of the native.

The base-pairing ability of the base pair-mimic nucleosides

Nakano, S.-i., Uenishi, K., Fujii, M., Sugimoto, N. — 2007-11-20

The deoxyadenosine derivative tethering the phenyl group at N6 of deoxyadenosine (A^phe) was previously found to have a property to stack strongly with adjacent nucleotide bases in a DNA duplex. On the other hand, it was also demonstrated that DNA polymerases selectively incorporated dTTP opposite A^phe in a template DNA strand. These observations suggest that the conformation of A^phe in solution differs from that during the DNA polymerase reaction. Here, the chemical modifications of thymine bases in a DNA duplex by KMnO₄ and CMCT (1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate) were examined, and it was revealed that the thymine base opposite A^phe was efficiently flipped out of the DNA helix as much as that in a single-stranded DNA.

Effect of poly(L-lysine)-g-dextran copolymers on DNA hybridization

Wu, L., Shimada, N., Kano, A., Maruyama, A. — 2007-11-20

We have focused on design of biomaterials that are capable of engineering nucleic acids folding. In this study, we explored the effect of poly(L-lysine)-g-dextran, PLL-g-Dex, copolymer on hybridization kinetics of DNA. The hybridization kinetics was studied by using a fluorescence resonance energy transfer (FRET) assay in the absence and presence of the copolymer. The copolymer at nano molar concentration significantly accelerated the hybridization rate over 200-fold under physiologically relevant ionic conditions.

Structural and physicochemical features on the metal-mediated base pairs

2007-11-20

The chemical structure of the mercury-mediated T-T pair (T-Hg^II-T) was determined with ¹⁵N NMR spectroscopy. In order to determine the chemical structure of the T-Hg^II-T pair, ¹⁵N-¹⁵N J-coupling across a metal center (2JNN) was employed. Notably, this is the first observation of ²J_NN in a biological macromolecule (DNA duplex). This pairing mode was found to be a irregular metal ion-binding mode for DNA and RNA molecules, in which imino proton-metal exchange processes are included. Accordingly, ²J_NN is highly important for the determination of the chemical structures of metal-mediated base pairs.

Interactions with RNA/DNA of proteins involved in the regulation of transcription, translation and telomere elongation

2007-11-20

Interactions with DNA and RNA of three different proteins involved in the regulation of (1) transcription, (2) translation, and (3) telomere elongation were examined by NMR. In the first case, the combination of structural determination, dynamical analysis on the basis of relaxation data and identification of interactive surface for wild and phosphorylation-mimicking mutant proteins has given the insight on the increase of DNA-binding affinity through phosphorylation of the protein. In the second case, the arrangement of two tandem domains interacting with RNA has been determined with residual dipolar couplings and paramagnetic relaxation enhancement, which has given the idea on how the two tandem domains recognize the target RNA. In the third case, simultaneous binding of the other two tandem domains to both DNA and RNA has been analyzed with chemical shift perturbation analysis. The result has suggested that the protein composed of two tandem domains can recruit telomerase to telomere DNA.

Spectroscopic studies on a novel intramolecular hydrogen bond within the (6 4) photoproduct

Yamamoto, J., Tanaka, Y., Hitomi, K., Getzoff, E. D., Iwai, S. — 2007-11-20

The (6–4) photoproduct, which is one of major UVinduced lesions formed between adjacent pyrimidine bases in DNA, was characterized by using ¹⁵N NMR, fluorescence emission, and UV/VIS absorption at various pH values. From these experiments, we identified a novel intramolecular hydrogen bond between the hydroxyl group at the C5 position of the 5' component and the N3 of the 3’ pyrimidone ring within the (6–4) photoproduct.

Chemical Biology of DNA Polymerases: From Selectivity to New Functions

Marx, A., Summerer, D., Sauter, K. B. M., Gloeckner, C., Rudinger, N. Z. — 2007-11-20

DNA polymerases are involved in all DNA synthesis occurring in nature. Furthermore, DNA polymerases are the workhorses in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing and nucleic acids based diagnostics.

In order to identify DNA polymerase mutants with altered properties, we set up an efficient high through put setup to rapidly screen libraries of DNA polymerase mutants in automated parallel fashion and identified entities with significantly increased selectivity. Furthermore, our results indicate a rational to generally increase DNA polymerase selectivity as we demonstrate for several enzymes from different DNA polymerase families.

Additionally, we show that the generation of a new DNA polymerase function is achievable through iterative screening of small libraries of DNA polymerase derived by randomization of the respective genes. We demonstrate that the identified mutants find immediate applications and provide the basis for the development of new means for diagnostic technologies.

The Chemistry and Biology of RNA editing by Adenosine Deaminases

Beal, P. A., Maydanovych, O., Pokharel, S. — 2007-11-20

Deamination of adenosines within mRNAs catalyzed by ADAR enzymes generates inosines at the corresponding nucleotide positions. Since inosine is decoded as guanosine, this reaction can lead to codon changes and the introduction of amino acids into a gene product not encoded in the gene. Translation of the different coding strands created by this process leads to protein structural diversity in the parent organism and is necessary for nervous system function in metazoa. The basis for selective editing of adenosines within certain codons is not well understood at the structural/biochemical level. Here we describe the use of synthetic nucleoside analogs incorporated into RNA editing substrates via the protected phosphoramidites to define aspects of the editing reaction mechanism and to carry out mechanism-based trapping of ADAR-RNA complexes. In addition, a high-throughput screen has been developed capable of rapidly identifying functional editing systems.

Searching study for the suitable ferrocenylnaphthalene diimide derivative in the electrochemical telomerase assay

Sato, S., Ohtsuka, K., Takenaka, S. — 2007-11-20

Interaction of ferrocenylnaphthalene diimide 1 with the tetraplex oligonucleotide, AGGG(TTAGGG)3, which is a part of human telomere sequence, was studied in 0.1 M AcONa-AcOH (pH 5.5) containing 0.1 M NaCl coupled with 22-meric single and double stranded oligonucleotides using spectrophotometric titration experiment to search for the more suitable tetraplex DNA-binding ligand to achieve the electrochemical telomerase assay. CD spectra and polyacrylamide gel electrophoresis revealed that this tetraplex oligonucleotide kept to the single conformational structure of basket type G-quadruplex under these conditions. Scatchard analysis showed that 1 can bind to the tetraplex oligonucleotide with the binding constant of 10⁵ M^–1 order, which was the highest value among the other oligonucleotides. Binding number of 1 for these oligonucleotides were ca. 2, 11, and 3 for tetraplex, double stranded, and single stranded oligonucleotides, respectively. These values were reasonable when considering with the binding mode of 1 as a threading intercalator.

Application of on-chip capillary electrophoresis to cell-free preparation of recombinant DNA

Nojima, T., Kaneda, S., Fujii, T. — 2007-11-20

An on-chip capillary electrophoresis-based DNA collection was applied to the isolation of target DNA species from a DNA mixture generated by a polymerase chain reaction (PCR), whose starting material was a ligation mixture of an insert and an expression vector. The collected DNA was then amplified by PCR and properly worked as template DNA in a coupled cell-free transcription/translation system. These results demonstrateed that total operation in standard genetic engineering can be performed in a cell-free condition.

Binding Effect of the Antitumor Active Dirhodium (II) Compounds to Plasmid DNA

Rahman, Md. M., Yasuda, H., Takashima, K., Mizuno, A. — 2007-11-20

Binding effect of the antitumor dirhodium (II) compounds to the plasmid pUC19 DNA has been studied under different molar ratio of Rh (II) compounds to base pair of pUC19 DNA (Rf) and time. The electrophoresis results indicate that Rh binding affect the DNA conformation and therefore, Rh-DNA migrated in the different position comparing with native DNA. At high value of Rf ICP-MS (Inductively Coupled Plasma Mass Spectrometry) measurement confirmed that 46% of Rh binds to DNA. In vitro result shows that DNA synthesis was inhibited by the Rh binding.

Gene regulation by decoy approach (II): Development of photo-cross-linked oligonucleotides duplex as a decoy DNA for estrogen receptor

Yamayoshi, A., Shimazu, N., Higuchi, M., Kobori, A., Murakami, A. — 2007-11-20

We have developed a photo-cross-linked oligonucleotide (clip-ODN) for a novel type of gene regulator molecule. Here, we examined the ability of the clip-ODN as a decoy DNA on regulation of the transcriptional activity of estrogen receptor (ER). A photo–cross-linking reagent, 4,5',8-[4'-(aminoethyl-amino) methyl]-trimethylpsoralen (aeAMT) was conjugated with an ODN at the 5'-end, and the aeAMT was cross-linked with the thymine residue of the complementary oligonucleotide upon UVA irradiation (365nm). The clip-ODN drastically inhibited the proliferation of breast cancer cell line (MCF-7) than non-cliped one in a sequence specific manner. This finding revealed that photo-cross-linking of double stranded ODN improve the regulatory ability as a decoy DNA, and clip-ODN may be a valuable tool in gene therapy protocols for inhibiting breast cancer cells’ proliferation.

Reversible regulation of binding between a photoresponsive peptide and its RNA aptamer

Hayashi, G., Hagihara, M., Dohno, C., Nakatani, K. — 2007-11-20

It will be important for biologists to artificially and reversibly control gene expression in vivo through the interaction of RNA and small molecules. In this symposium, we report that RNA aptamers obtained from in vitro selection in which a photoresponsive short peptide containing the azobenzene moiety with flanking arginine residues on both sides as a ligand provided reversible binding to the ligand peptide immobilized onto the gold surface. We designed and synthesized a photoresponsive short peptide that can interact with RNA, can convert its conformation reversibly by photoirradiation, and can be produced on a large scale for in vitro selection. The RNA pool contained N70 random sequences, and after the eighth cycle we identified RNA aptamers showing the Kd of about a few µM. A surface plasmon resonance (SPR) experiment revealed that RNA aptamers could bind to the transisomer of the peptide immobilized on the gold surface, but not to the cis-peptide isomerized by photoirradiation with 360 nm light to the gold surface. The SPR signals were recovered after photoirradiation with 430 nm light leading to isomerization of the peptide from cis to trans.

Structure-based Design of Fluorescent Biosensors from Ribonucleopeptide Complexes

Hayashi, H., Inoue, M., Morii, T. — 2007-11-20

Fluorescent biosensors are crucial tools to facilitate sensitive detection of small molecules. However, construction of fluorescent biosensors with desired characteristics, that is, detection wavelengths and concentration ranges for ligand detection, from macromolecular receptors is not a straightforward task. Previously, we reported a modular strategy (Hagihara, et al., J. Am. Chem. Soc., 2006, 128, 12932) for constructing fluorescent ATP sensors from ribonucleopeptide (RNP) complexes. These RNP sensors had a variety of emission wavelengths and/or responding ligand concentration ranges. The modular strategy allows choosing a fluorescent RNP sensor with appropriate optical properties, but does not allow us to design optimal properties of fluorescent RNP sensors. Here we report a design strategy to optimize the response of fluorescent RNP sensors based on the secondary structural analyses of ATP-binding RNPs. We analyzed the RNA secondary structure of RNP sensors and examined the relationship between the secondary structure and fluorescent characteristics. We have discovered that fluorescent ATP sensors with high value of I/I0 ratio tend to contain interior loop and to record low fluorescence intensity in the absence of ATP. Indeed, ATP sensors, which was inserted sequence that construct an interior loop of ATP sensor with large fluorescence intensity change, were induced increase of fluorescent intensity change. This result suggests that use of the secondary structural elements would allow a rational functional design of RNP.

SNPs typing based on the formation of fluorescent signaling DNA aptamers which bind to bile acids

Kato, T., Kamoto, S., Hyuga, M., Karube, I. — 2007-11-20

A simple and rapid method for the detection of single nucleotide polymorphisms (SNPs) is essential for the development of personalized medicine because SNPs correlate with some diseases and side effects of some drugs. Here we report a new method for the fluorescent detection of single nucleotide mutations that is based on the formation of cholic-acid binding DNA aptamers which form fully matched three-way junctions.

Aptameric enzyme subunit for homogeneous protein sensing

Yoshida, W., Sode, K., Ikebukuro, K. — 2007-11-20

We have developed an aptameric enzyme subunit (AES) for protein sensing. AES is composed of an enzyme-inhibiting aptamer and a target molecule-binding aptamer and it can allosterically control the corresponding enzyme activity by binding target molecules. Here, we applied AES to immunoglobulin E (IgE) sensing. We split the thrombin-inhibiting aptamer into two oligonucleotides and then connected IgE-binding aptamer and its complementary strand to the split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin enzymatic activity and it was decreased in the presence of IgE. We were able to detect IgE by using the AES in homogeneous assay and detection limit was 250 nM.

Animated Crystallography of Genetic Code Translation

Tomita, K., Numata, T., Fukai, T., Nureki, S., Ishitani, R., Nureki, O. — 2007-11-20

Accurate genetic code translation is achieved by accumulation of hyper-specific chemical reactions. Transfer RNA (tRNA) acts as an adaptor molecule to link the genetic code (in messenger RNA) to a specific amino acid. tRNA itself has no ability to specifically bind with the cognate amino acid, while aminoacyltRNA synthetases (aaRSs) strictly recognize their cognate tRNA and amino acid, to synthesize a correctlypaired aminoacyl-tRNA, a substrate for ribosome. tRNA is initially transcribed by RNA polymerase as a precursor RNA with long extensions at the 5' and 3' terminus. Maturation of tRNA to a functional RNA requires processing of the extensional sequences and post-transcriptional chemical modifications, which ensures the specific aminoacylation of tRNA by the cognate aaRS. We have solved the crystal structure of the tRNA-maturating enzymes in a complex with tRNA (precursor) to especially elucidate the sophisticated mechanism of their highly specific chemical reactions. Our goal is to provide the animated crystallography or "movie" of the dynamic processes in the genetic code translation by capturing snapshots of the chemical reaction steps by X-ray crystallography.

The chemistry of transcription through damaged DNA and of translesion synthesis at atomic resolution

Carell, T., Cramer, P., Hopfner, K.-P. — 2007-11-20

Small regulatory RNAs: biogenesis & functions

2007-11-20

Massively Parallel Synthetic Sequencing of DNA on Clonal Arrays

Balasubramanian, S. — 2007-11-20

Design, synthesis, and biological activity of carbocyclic analogues of cyclic ADP-ribose, a Ca2+-mobilizing second messenger

Shuto, S. — 2007-11-20

An efficient method for the total synthesis of cyclic ADP-ribose (cADPR, 1) analogues was established. In this procedure, formation of the characteristic 18membered ring was key step, which was achieved by the AgNO3-or I2-promoted condensation with the phenylthiophosphate-type substrate forming an intramolecular pyrophosphate linkage. Using this method, a variety of carbocyclic analogues of cADPR have been synthesized to investigate the structure-activity-relationship, where cyclic ADP-carbocyclic-ribose (2) was identified as a stable and cell-type selective cADPR agonist.

In vivo antisense activity of ENA(R) oligonucleotides targeting PTP1B mRNA in comparison of that of 2'-MOE-modified oligonucleotides

Koizumi, M., Takagi-Sato, M., Okuyama, R., Araki, K., Sun, W., Nakai, D. — 2007-11-20

The 2'-0-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide ISIS113715, which targets protein-tyrosine phosphatase IB (PTP1B) mRNA, increases insulin sensitivity and normalizes plasma glucose levels in diabetic ob/ob and db/db mice. In the present study, the efficacy of the isosequential 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified oligonucleotide ENA-1 was compared with that of ISIS113715 in order to further improve the down-regulation of PTP1B in db/db mice. Intraperitoneal administration of ENA-1 more effectively decreased the plasma glucose levels in db/db mice than ISIS113715. Moreover, ENA-1 decreased the expression of PTP1B in the liver and fat of db/db mice more effectively than ISIS113715. These data indicate that ENA modifications enhance the ability of antisense oligonucleotides and make them superior to second-generation 2'-MOE modifications.

We would like to thank to Drs. Shinya Tsutsumi and Kenji Kawai for the T_m measurement and autoradiography experiments. ENA is a registered trademark of Mitsubishi-Kagaku Foods Corporation.

Inhibition of bcl-xLexpression by antisense oligonucleotides containing various bridged nucleic acids (BNAs)

Roongjang, S., Takahashi, K., Obika, S., Imanishi, T. — 2007-11-20

Recently, we described the antisense activity of 2’,4’-BNA oligonucleotides in living cells.¹ Here, we examine the antisense effect of 2’,4’-BNA, 2’,4’-^COC and 3’-amino-2’,4’-BNA oligonucleotides targeting the bcl-xLgene. The results showed that while S-oligo had a slightly inhibiting effect on bcl-xLexpression and natural DNA had no effect, an antisense oligonucleotide (AON) containing six 2’,4’-^COC nucleotides reduced the bcl-xLmRNA level to 64% of the untreated level. Interestingly, several types of AON—those containing nine 2’,4’-BNA^COC nucleotides, 2’,4’-BNA and 3’-amino-2’,4’-BNA— completely inhibited expression of the target gene. These data show that, compared with the corresponding natural DNA and S-oligo, BNA-based AONs are efficient inhibitors of bcl-xLexpression.

Insitugeneration of aminoacyltRNAs assisted by ribozymes in translation apparatus

Ohuchi, M., Murakami, H., Suga, H. — 2007-11-20

Flexizymes are artificial RNA catalysts that enable us to readily prepare aminoacyltRNAs with a variety of amino acid and tRNA kinds. On the other hand, because their flexibility feature lacking high specificities toward amino acids and tRNAs, the insituaminoacylation in a translation apparatus have not been able to executed. We here present a novel strategy to overcome this specificity problem to tRNA using a cisacting flexizyme–tRNA construct, called a catalytic precursor tRNA, combining with a naturally occurring ribozyme, ribonuclease P (RNase P). In this coupling system of two RNA enzymes, selfaminoacylation occurs on the catalytic precursor tRNA for specific charging of amino acids at the 3'end of the tRNA domain in the presence of the cognate amino acid substrates. Subsequently, the aminoacylated catalytic precursor tRNA is specifically cleaved at the 5'linker region of the tRNA domain, giving the mature aminoacyltRNA. Most importantly, the generated flexizyme does not function intransto tRNAs present in the translation apparatus, indicating that this tworibozyme coupling system would potentially act as an orthogonal aminoacylation system in the translation apparatus.

Versatility of borane nucleic acids mimics for coding, decoding and modulating genetic information

Shaw, B. R. — 2007-11-20

This presentation will focus on the targeted regulation of gene expression, effective siRNA silencing with boranophosphates, and suppression of drugresistant reverse transcriptase by boranophosphate nucleotide analogues.

Chemical synthesis and properties of stereoregulated phosphorothioate RNAs

Wada, T., Kondo, T., Fujiwara, S., Sato, T., Oka, N. — 2007-11-20

Stereoregulated oligoribonucleoside phosphorothioates were synthesized by the use of 2’-O-TBDMS-protected ribonucleoside 3’-O-oxazaphospholidine derivatives as monomers and N-(cyanomethyl)ammonium salts as activators. Diastereoselectivity of the condensation reaction was found to be highly dependent on the substituent groups of the oxazaphospholidine ring as well as the structure of the activators. By the use of the optimized oxazaphospholidine monomers and activators, stereoregulated oligoribonucleoside phosphorothioates containing four kinds of nucleobases were synthesized in good yields. Hybridization properties of thus obtained oligomers with the complementary RNAs were evaluated by the UV melting experiments.

Fluorosensor proteins to detect specific DNA sequences in living bacteria

Waki, S., Yoshitake, K., Iwasaki, R., Ueda, H. — 2007-11-20

While there are many hybridization-based DNA sensors, few of them can detect native double-stranded DNA, which is most commonly found in physiological conditions. Here we made novel fluorosensor proteins comprised of a pair of two zinc fingers tethered with an N-terminal dimerization motif and a C-terminal yellow fluorescent protein fragment (split eYFP) to detect specific DNA sequence in a living bacteria. When E. coli Top10 cells harboring the plasmid encoding the fusion proteins and a test plasmid encoding target DNA sequence were induced for the protein expression, significant increase in fluorescence was observed, compared with the strain harboring a test plasmid without target DNA sequence.

Location of the triplex DNA-binding domain of Saccharomyces cerevisiae Stm1 protein

Katayama, T., Inoue, N., Torigoe, H. — 2007-11-20

Saccharomyces cerevisiae Stm1 protein (273 amino acids) is a purine motif triplex DNA-binding protein. Here, to examine the location of the triplex DNAbinding domain of Stm1 protein, we analyzed the interaction between the purine motif triplex and a series of truncated Stm1 proteins by electrophoretic mobility shift assay. Stm(1-113) (amino acids 1-113) was able to bind with the purine motif triplex. Smaller regions in Stm(1-113), Stm(1-54) (amino acids 1-54) and Stm(55-113) (amino acids 55-113), were unable to bind with the purine motif triplex. Although Stm(1-113) has the ability to bind with the purine motif triplex, it was unable to bind with the duplex. We conclude that Stm(1-113) is the minimal domain to specifically bind with the purine motif triplex.

Food Restriction in Pregnant Mice Can Induce Changes in Histone Modifications and Suppress Gene Expression in Fetus

2007-11-20

Biological responses to environmental effects are mediated through epigenetic changes such as chemical modifications of the histone tails and DNA (5-cytosine) methylation. We report that dietary protein restriction in pregnant mice can alter histone modifications in the promoter region of the Igf2 gene and cause a two-fold repression in promoter specific Igf2 transcription in the liver of the fetus. Suppression of Igf2 is accompanied with low birth weight of the pups born to the protein-restricted dams. Our results provide new information about the epigenetic aspects of early life programming and will improve our understanding about the developmental origins of adult diseases.

Photo inducible RNA interference using cell permeable protein carrier

Endoh, T., Sisido, M., Ohtsuki, T. — 2007-11-20

In this study, we constructed fluorescently labelled protein carrier constructed from cell-penetrating peptide (CPP) and RNA binding domain (RBD) for intracellular shRNA or siRNA delivery. The protein carrier specifically bound to an RNA cargo containing short extended sequence tag for protein binding, and internalized into CHO cells together with the RNA. Although the internalized protein/RNA complexes showed cytoplasmic punctuate distributions, they widely spread into cytosol by photo irradiation. Moreover, redistributed RNA induced gene silencing only within the photo irradiated area.

Short-step chemical synthesis of DNA by use of MMTrS group for protection of 5' hydroxyl group

Shiraishi, M., Utagawa, E., Ohkubo, A., Sekine, M., Seio, K. — 2007-11-20

4-methoxytrithylthio (MMTrS) group was applied for the appropriately protected four canonical nucleosides. We prepared the phosphoroamidite units by use of these nucleosides and developed the synthesis of oligodeoxynucleotides without any acidic treatment. Moreover, the new DNA synthesis protocol was applied to an automated DNA synthesizer for the synthesis of longer oligodeoxynucleotides.

Development of a new method for the synthesis of oligodeoxynucleotides by use of carbamoyl-type protecting groups

Ohkubo, A., Kasuya, R., Miyata, K., Taguchi, H., Seio, K., Sekine, M. — 2007-11-20

Previously, we observed thermal elimination of a quinolylcarbamoyl group from Nquinolylcarbamoylated nucleobase residues. In this paper, we examined the thermal stability of several carbamoyl groups on nucleobases and developed new carbamoyl-type protecting groups for base protection that can be removed by "heating". Particularly, the phenylsulfonylcarbamoyl group was found to be rapidly removed from three kinds of nucleobases, cytosine, adenine, and guanine bases. Moreover, we could carry out the synthesis of oligodeoxynucleotides by using the thermolytic protecting group for base protection.

Nucleoside H-boranophosphonates: Synthesis and properties of a new class of nucleotide analogs

Higashida, R., Kawanaka, T., Oka, N., Wada, T. — 2007-11-20

Nucleoside H-boranophosphonates were synthesized via the condensation reactions of appropriately protected nucleosides with monopyridinium Hboranophosphonate. The condensation reactions gave only the mono-esterified products under the optimized conditions without formation of di-esterified byproducts. Deprotection of the condensation products was achieved under basic conditions to afford the fully-deprotected nucleoside H-boranophosphonates in excellent yields.

Synthesis of a guanosine phosphoramidite derivative containing a photocleavable protecting group at the O6 position via regioselective protection of the 2'-hydroxy group with a TBDMS group

Iwase, R., Toyama, T. — 2007-11-20

To construct modified RNA that form A-type duplex with photo-irradiation, a photocleavable a-methyl-2-nitropiperonyl (MeNP) group was introduced at O⁶ position of guanosine. A guanosine phosphoramidite derivative containing the MeNP group was synthesized via regioselective 2'-O-protection of 3',5'-O-di(t-butyl)silanediylguanosine with TBDMS group. The MeNP group was found to be stable under conditions of solid-phase synthesis of RNA. The MeNP group was also found to be removable by UV irradiation at wavelength of 365 nm for 10 min.