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We have developed and applied a computational strategy to increase the affinity of fullerene-based inhibitors of the HIV protease. The result is a approximately 50-fold increase in affinity from previously tested fullerene compounds. The strategy is based on the design of derivatives which may potentially increase hydrophobic desolvation upon complex formation, followed by the docking of the hypothetical derivatives into the HIV protease active site and assessment of the model complexes so formed. The model complexes are generated by the program DOCK and then analyzed for desolvated hydrophobic surface. The amount of hydrophobic surface desolvated was compared with a previously tested compound, and if this amount was significantly greater, it was selected as a target. Using this approach, two targets were identified and synthesized, using two different synthetic approaches: a diphenyl C60 alcohol (5) based on a cyclopropyl derivative of Bingel (Chem.Ber. 1993, 126, 1957-1959) and a diisopropyl cyclohexyl C60 alcohol (4a) as synthesized by Ganapathi et al. (J. Org.Chem. 1995, 60, 2954-2955). Both showed tighter binding than the originally tested compound (diphenethylaminosuccinate methano-C60, Ki = 5 microM) with Ki values of 103 and 150 nM, respectively. In addition to demonstrating the utility of this approach, it shows that simple modification of fullerenes can result in high-affinity ligands of the HIV protease, for which they are highly complementary in structure and chemical nature.

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The process of RNA interference can be controlled with light. On the following pages, S. H. Friedman and co-workers demonstrate that siRNAs modified with photolabile groups have reduced ability to effect RNA interference until irradiation deprotects them and releases fully active siRNA.

The spacing, timing, and amount of gene expression are crucial for a range of biological processes, including development. For this reason, there have been many attempts to bring gene expression under the control of light. We have previously shown that RNA interference (RNAi) can be controlled with light through the use of siRNA and dsRNA that have their terminal phosphates modified with the dimethoxy nitro phenyl ethyl (DMNPE) group. Upon irradiation, these groups photolyze and release native RNA. The main problem with light activated RNA interference (LARI) to date is that the groups used only partially block RNA interference prior to irradiation, thus limiting the utility of the approach. Here, we describe a new photocleavable group, cyclo-dodecyl DMNPE (CD-DMNPE), designed to completely block the interaction of duplexes with the cellular machinery responsible for RNA interference prior to irradiation. This allowed us to switch from normal to a near complete reduction in gene expression using light, and to construct well-defined patterns of gene expression in cell monolayers. Because this approach is built on the RNA interference pathway, it benefits from the ability to quickly identify duplexes that are effective at low or subnanomolar concentrations. In addition, it allows for the targeting of endogenous genes without additional genetic manipulation. Finally, because of the regiospecificity of CD-DMNPE, it allows a standard duplex to be quickly modified in a single step. The combination of its efficacy and ease of application will allow for the facile control of the spacing, timing, and degree of gene expression in a range of biological systems.

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.

Bringing RNA interference (RNAi) under the control of light will allow the spacing, timing, and degree of gene expression to be controlled. We have previously shown that RNAi by small interfering (si) RNA can be modulated through randomly incorporated photolabile groups. Our and others interest is to find key locations on siRNA that can completely block RNAi until irradiation releases completely active siRNA. Some literature suggests that the 5' phosphate of the antisense strand of siRNA cannot be modified without completely blocking RNAi. We have examined this site as a potential switch for light control of RNAi and present evidence that siRNA modified at the 5' antisense phosphate can still cause RNAi, although not at the level effected by fully native siRNA. This contrasts with results from the literature, which suggest that modification of the 5' antisense phosphate will completely abrogate RNAi in siRNA. We have used mass spectrometry to identify and quantitate possible impurities that may be responsible for residual RNAi and show that they are present at 1% or less. Our results suggest that there is an inherent tolerance of the RNAi machinery toward modification of the 5' antisense phosphate.