Chemistry for gene silencing: 4'-modified and 2'-fluorinated nucleosides and oligonucleotides

A series of studies on 2'-fluorinated and 4'-chalcogen-modified nucleic acids is described, mainly directed toward the development of better gene silencing therapeutics.;The stability of 2'F-ANA to acid-mediated hydrolysis was compared to that of DNA and RNA. Several phosphodiester (PO) or phosphorothioate (PS) 2'F-ANA sequences were incubated at pH ∼1.2, and virtually no cleavage was observed after 2 days. In contrast, rapid degradation was observed for DNA (t1/2 = minutes) and RNA (t1/2 = hours (PO) or days (PS)). The nuclease-catalyzed hydrolysis of 2'F-ANA was also explored in detail. One diastereomer of the PS-2'F-ANA linkage was much more vulnerable to enzymatic cleavage than the other, which is parallel to the properties observed for PS-DNA. We also show that the nuclease stability induced by 2'F-ANA depends on the oligonucleotide sequence.;An improved synthesis of 2'-deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) is described. Participation of the 3'-O-benzoyl protecting group in the thiosugar precursor influenced the stereochemistry of the N-glycosylation reaction in nonpolar solvents, permitting a higher beta:alpha ratio than previously observed for similar Lewis acid-catalyzed glycosylations. The nucleoside adopted a predominantly northern conformation, in contrast to 2'-deoxy-2'-fluoro-5-methylarabinouridine (FMAU), which adopts a predominantly southeast conformation.;The synthesis of oligonucleotides containing 2'-deoxy-2'-fluoro-4'-thioarabinonucleotides is then described. 18-mer antisense oligonucleotides (AON) containing 4'S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern (O4'-endo) conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD) and UV binding studies. Replacement of the 4'-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON•RNA as well as AON•DNA duplexes. 2'-Deoxy-2'-fluoro-4'-thioarabinouridine (4'S-FAU) was incorporated into siRNA and the resulting siRNA duplexes were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2'F-ANA was demonstrated. This synergy inspired us to combine other northern nucleosides with 2'F-ANA, and we discovered that combination of 2'F-RNA and 2'F-ANA leads to fully modified siRNA duplexes that are more potent than the control siRNAs. This is one of only a few chemistries allowing fully modified siRNAs with increased potency. Several related patterns of chemical modification were used to modify two siRNA sequences targeting firefly luciferase.;NMR/MD and osmotic stressing were used to compare the structure and hydration of 10-bp 2'F-ANA•RNA, ANA•RNA and DNA•RNA duplexes. The 2'F-ANA and ANA strands both featured sugars that pucker in the east (O4'- endo) conformation, as previously observed for hairpin structures containing hybrid stems. Osmotic stressing suggested that the 2'F-ANA•RNA duplex liberated fewer molecules of water upon melting than did ANA•RNA, which may give the former an entropic advantage that contributes to its far greater thermal stability.;The first synthesis of oligonucleotides containing 4'-selenium-modified ribonucleotides (4'-Se-rN) is described. Four sequences containing the 4'-selenoribonucleotide were successfully synthesized, and compared with DNA and RNA oligonucleotides containing a dT, rT or LNA insert in place of the 4'-Se-rT. The 4'-Se-rT behaved more like rT than dT in its effects on binding affinity, despite the DNA-like structure previously observed for the nucleoside. Incorporation of 4'-Se-rT into A-RNA and hybrid duplexes led to increased binding affinity, while incorporation into B-DNA destabilized the duplex to the same extent as an rT nucleotide.