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

• Published in: Nucleic acids research 2021-12, Vol.49 (21), p.12069-12088
• Main Authors: Yamada, Ken, Hildebrand, Samuel, Davis, Sarah M, Miller, Rachael, Conroy, Faith, Sapp, Ellen, Caiazzi, Jillian, Alterman, Julia F, Roux, Loic, Echeverria, Dimas, Hassler, Matthew R, Pfister, Edith L, DiFiglia, Marian, Aronin, Neil, Khvorova, Anastasia
• Format: Article
• Language: English
• Subjects: Alleles; Chemical Biology and Nucleic Acid Chemistry; Humans; Huntington Disease - genetics; Oligonucleotides - metabolism; Organophosphonates; RNA, Small Interfering - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab1126

Abstract 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 (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-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, iE-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 iE-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. Graphical Abstract Graphical Abstract Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.