Synthesis, chirality-dependent conformational and biological properties of siRNAs containing 5′-(R)- and 5′-(S)-C-methyl-guanosine

Abstract

Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5′-C-methyl pyrimidine nucleotides also modified at the 2′ position of the sugar. Here, we describe the synthesis of 2′-position unmodified 5′-(R)- and 5′-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5′ exonuclease and the (S) isomer provided protection from 3′ exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5′ triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5′ and 3′ exonuclease oligonucleotide complexes with 5′-(R)- and 5′-(S)-C-methyl substituents attached to the 5′- and 3′-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5′-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5′-C-methylation on endoribonuclease activity has been explained.

The current landscape of nucleic acid therapeutics

“…Careful siRNA sequence selection, especially of seed regions, reduces the risk of hybridization-dependent off-target effects. Additional siRNA modifications, including glycol nucleic acid substitution in the seed region (ESC+) or altriol nucleic acid residues, might be implemented in future conjugates to further boost RNAi potency and curtail adverse effects 51,52 Chirality-dependent properties could also offer additional functionalities 53 . Balancing further chemical modifications and compatibility with RNAi machinery will be vital to the next generation of siRNA therapeutics.…”

Photochemical Internalization of siRNA for Cancer Therapy

“…The first FDA-approved siRNA therapeutic, Patisiran, is composed of multicomponent lipid nanoparticles (LNP) encapsulating partially chemically modified siRNA, in which some of the nucleotides are chemically modified at 2′-OMe. These chemical modifications reduce the nuclease degradation and innate immune system stimulation, while LNP provides the liver-specific delivery of siRNA via apolipoprotein E (ApoE) receptor endocytosis aside from nuclease protection [ 39 ].…”

Synthesis, gene silencing activity, thermal stability, and serum stability of siRNA containing four (S)-5′-C-aminopropyl-2′-O-methylnucleosides (A, adenosine; U, uridine; G, guanosine; and C, cytidine)

“…Recently, Manoharan et al reported the synthesis and properties of C4 0 and C5 0 -modied nucleosides, such as the 4 0 -C-methoxy-modied nucleosides, (R)-, or (S)-5 0 -C-methyl-modied nucleosides. [14][15][16][17][18][19] These nucleosides enhanced the nuclease resistance of oligonucleotides, compared with C2 0 -modied nucleosides without thermally destabilizing RNA duplexes and inhibiting gene silencing activity. Moreover, (R)or (S)-5 0 -C-methylnucleosides improved the stability of oligonucleotides to snake venom phosphodiesterase (SVPD), compared with 4 0 -C-methoxy-nucleoside.…”