Design and synthesis of amide-linked ribonucleic acids for potential application in RNA interference

The ongoing research has since revealed that RNA is not just a simple intermediate in flow of genetic information but also serves as a key catalyst in most fundamental biological processes. RNA also plays a central role in controlling gene expression in eukaryotes and prokaryotes. Antisense oligonucleotide is a tool used to inhibit gene expression with potential that may be used in studying gene function and gene therapy. Various chemical modifications have been used to enhance nuclease resistance to antisense oligonucleotides.;RNA interference is very powerful technique that can be used to silence gene expression in a sequence-specific manner. Synthetic short interfering RNAs, with complementary gene sequence to mRNA can be introduced to the cells and incorporated in the RISC complex to degrade the target mRNA. The discovery of RNAi reinvigorated research interest into chemical modification of short synthetic oligonucleotides to stabilize and improve the efficacy of siRNAs and advance knowledge on the general mechanism of RNAi. Similar modifications used on antisense oligonucleotides have also been used in siRNAs. Unmodified phosphodiester backbone is catalytically cleaved at the oxygen-phosphate bond by nucleases thus degrading the RNA. Furthermore, phosphodiester backbone is negatively charged which creates difficulty in crossing the negatively charged lipids. Modification at the phosphodiester linkage has been explored ranging from single atom replacement to where the entire backbone is replaced with neutral and non-ionic linkage. Amides as neutral and hydrophobic internucleotide linkage in both DNA and RNA are excellent mimics of the natural phosphodiester. Previous studies have shown that amides are interesting hydrophobic, non-ionic modifications that may provide high nuclease resistance and suggest a hypothesis that siRNAs may tolerate even more extensive consecutive amide modification. Testing the above hypothesis requires efficient large-scale synthesis of enantiomerically pure monomeric analogs of C3'-homologated carboxylic acids. Amide modified RNA could be prepared using reasonably straightforward solid-phase peptide-type coupling chemistry. The synthetic challenge has been the preparation of monomeric units with C3'-homologated nucleosides required for the peptide coupling chemistry. The main hurdle has been the synthesis of efficient monomeric building blocks for introduction of consecutive amide linkages on large scale and ultimately incorporated to solid-phase synthesis.;In this thesis, we have synthesized monomeric building blocks required for the solid-phase amide-linked synthesis. We optimized a synthetic procedure used to produce highly modified nucleoside amino acid from simple carbohydrate. The modified monomers enabled us to manually synthesize consecutive amide linkages and incorporated into the automated phosphoramidite synthesis. The optimized manual peptide-type coupling gave an average coupling yield of 88 - 90%. The purified siRNAs containing consecutive amide linkages near the 5'-end and short 10-mer sequence with three amide linkages at the middle of the sequence were subjected to mass spectroscopy to ensure the full length.;The biophysical properties of the three consecutive amide modifications in the 10-mer duplex were investigated. The techniques used in this work included UV thermal melting, secondary structure determination and osmotic stress experiments. Melting temperature experiments revealed a slight destabilizing effect (∼3.5 ˚C/ modification) when compared to the unmodified control duplex. The thermodynamic DeltaH parameter was extracted from three independent experiments (van't Hoff analysis, calorimetric technique and concentration dependent experiments) and used to determine the hydration properties. The observed increase in melting temperatures upon increase in total concentration of the duplex revealed that there was duplex formation as opposed to a hairpin. The solution NMR studies were used to verify the duplex secondary structure through base-pairing patterns. The conformational equilibria information also revealed the duplex structure as opposed to the hairpin. Furthermore, the molecular model of consecutive amide linkage annealed to unmodified control showed that the A-type geometry of RNA helix was not significantly changed. Circular dichroism experiments further illustrated that the three consecutive amide modifications exhibited the standard A-form double helix similar to the unmodified control. The osmotic stress experiments indicated that the introduction of three consecutive amide modifications does not significantly disturb the hydration of RNA. Finally, the effect of consecutive amide modifications on RNAi activity was tested using passenger strand of siRNA targeting the Cyclophilin B (PPIB) mRNA. Our preliminary biological activity of siRNAs containing two and three consecutive amide modifications near the 5'-end in the passenger strand indicated that the modification is compatible within the RNAi machinery. We observed that the two and three consecutive amide modification on sense strand caused very little change in the siRNA silencing activity. To the best of our knowledge, this is the first report involving consecutive amide modification within the internal positions of siRNAs.