Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (E-i-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for E-i-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the E-i-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of E-i-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, E-i-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the E-i-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.