Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Light Activated RNA Interference Using Photo-cleavable siRNA
My lab is bringing RNA interference under the control of light, by modifying siRNA and double stranded siRNA precursors (dsRNA) with photo-labile groups. The long term goal of this work is to allow the control of the spacing, timing and amount of expression of endogenous genes by varying the spacing, timing and amount of irradiation. The rationale behind the attachment of these photo-labile groups is that they will block the interaction of siRNA with RISC or dsRNA with Dicer prior to irradiation, thereby blocking RNA interference. Upon irradiation, native siRNA/dsRNA is released and RNA interference is induced. We have previously demonstrated that siRNA modified with the di-methoxy nitro-phenyl ethyl group (DMNPE) allows for modulation of RNA interference in a light dependent fashion. (See Angewandte Chemie 2005, Oligonucleotides 2007, Nature Protocols 2008) Because these groups do not completely block RNA interference prior to irradiation, we have extensively examined other systems with improved efficacy. Using MS/MS and other analytical techniques we have found that dsRNAs are preferentially modified with the DMNPE group on their terminal phosphates (as opposed to internal phosphates). In addition we have found that this allows for a more significant block of RNA interference prior to irradiation and makes modified dsRNA precursors much more effective at modulating RNA interference in a light dependent manner. The work that I will discuss combines an in-depth exploration of the chemistry of modification of nucleic acids by photolabile groups as well as the biological consequences of those modifications.
Division of Pharmaceutical Sciences