© 2008 Oxford University Press
This article appears in the following Nucleic Acid Symposium Series issue: Joint Symposium of the 18th International Roundtable on Nucleosides, Nucleotides and Nucleic Acids and the 35th International Symposium on Nucleic Acids Chemistry [View the issue table of contents]
Construction of a photo-switchable gene for turning on and off gene expression with light irradiation
1Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan and 2Core Research for Evolution Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
*: Hiroyuki Asanuma: asanuma{at}mol.nagoya-u.ac.jp.
Abstract
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.