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Nucleic Acids Research Advance Access published online on May 28, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp415
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chemistry and Synthetic Biology

Light-activated RNA interference using double-stranded siRNA precursors modified using a remarkable regiospecificity of diazo-based photolabile groups

Samit Shah, Piyush K. Jain, Ashish Kala, Dipu Karunakaran and Simon H. Friedman*

Division of Pharmaceutical Sciences, School of Pharmacy, 5005 Rockhill Road, University of Missouri-Kansas City, Kansas City, MO 64110, USA

*To whom correspondence should be addressed. Tel: +1 816 235 2224; Fax: +1 816 235 5779; Email: friedmans{at}umkc.edu

Received April 13, 2009. Revised May 4, 2009. Accepted May 5, 2009.

Diazo-based precursors of photolabile groups have been used extensively for modifying nucleic acids, with the intention of toggling biological processes with light. These processes include transcription, translation and RNA interference. In these cases, the photolabile groups have been typically depicted as modifying the phosphate backbone of RNA and DNA. In this work we find that these diazo-based reagents in fact react very poorly with backbone phosphates. Instead, they show a remarkable specificity for terminal phosphates and very modest modification of the nucleobases. Furthermore, the photo deprotection of these terminal modifications is shown to be much more facile than nucleobase modified sites. In this study we have characterized this regiospecificity using RNA duplexes and model nucleotides, analyzed using LC/MS/MS. We have also applied this understanding of the regio-specificity to our technique of light activated RNA interference (LARI). We examined 27-mer double-stranded precursors of siRNA (‘dsRNA’), and have modified them using the photo-cleavable di-methoxy nitro phenyl ethyl group (DMNPE) group. By incorporating terminal phosphates in the dsRNA, we are able to guide DMNPE to react at these terminal locations. These modified dsRNA duplexes show superior performance to our previously described DMNPE-modified siRNA, with the range of expression that can be toggled by light increasing by a factor of two.


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