Understanding the intricate interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of the gene silencing mechanism of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological (and potentially therapeutic) function.   In earlier studies of these interactions, the use of various chemically-modified siRNAs has been reported; however, few examples using nucleobase modifications exist.  Here we describe the synthesis and use of siRNAs bearing size-expanded RNA (xRNA) analogues as a novel set of steric probes.   xRNA nucleobases are expanded by 2.4 angstroms and retain canonical Watson-Crick base-pairing groups.  Their use in a dual-reporter luciferase assay in HeLa cells has revealed sites on the antisense strand where larger-than-natural nucleobases are tolerated, including positions where activity is higher than that of natural RNA.  Melting temperature and serum stability data provide further insights into RNAi-based gene silencing.

Investigating the protein-nucleic acid interactions of RISC using xRNA-containing siRNAs as steric probes in RNA interference

243rd National Meeting (2012)

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