BIOL 127 |
| Regulating gene expression with light-activated compounds has become increasingly important for elucidating gene function and developing oligonucleotide therapeutics. Hybridization of an antisense oligodeoxynucleotide (asODN) to a target mRNA can inhibit translation by sterically blocking the ribosome and/or recruiting endogenous ribonucleases. We set out to photomodulate asDNA/mRNA hybridization using a single photoactive moiety, in order to control gene expression most efficiently. With this goal, we designed and synthesized a series of light-activated DNA hairpins by covalently attaching a 20-mer asODN to a complementary sense strand through a heterobifunctional photocleavable linker, 1-(5-(N-maleimidomethyl)-2-nitrophenyl)ethanol N-hydroxysuccinimide ester. By varying the size of the hairpin loop, and the number of complementary base pairs and mismatches, the photoactive conjugates were stabilized by ~ 1-4 kcal/mole compared to the corresponding asODN/sODN duplexes. These differences in stability made it possible to regulate asODN/RNA duplex formation. In vitro RNase H assays showed 3-10-fold increases in RNA degradation upon photoactivation of the asODN/sODN hairpin. Experiments to photoregulate gene expression inside living cells will be described. |
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Chemistry and Metabolism
4:30 PM-6:30 PM, Tuesday, 12 September 2006 Moscone Center -- Hall D, Poster
Division of Biological Chemistry |