Influence of mismatched and bulged nucleotides on SNP-preferential RNase H cleavage of RNA-antisense gapmer heteroduplexes

Abstract

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.

Inhibition of off-target cleavage by RNase H using an artificial cationic oligosaccharide

“…ODAGal4 selectively inhibited the RNase H cleavage of mismatch RNA oligomers when using 9mer and 10mer DNAs. While there are several studies about improving mismatch discrimination using an appropriate design of oligonucleotides themselves, 7,[28][29][30] this study showed the possibility of improving mismatch discrimination by using an additive molecule. In addition, by combining this technology with a single methylphosphonate modification in a DNA strand, the range of application of ODAGal4 was extended to longer oligomers.…”

Reduction of Off-Target Effects of Gapmer Antisense Oligonucleotides by Oligonucleotide Extension

“…This may be because reduction of gene expression cannot be attributed solely on the strength of complementary binding between 18-mer ASOs and each gene. This indicates that, in addition to binding affinity, the off-target effect of gapmer ASOs is also related to other factors, such as the structure of RNA around the target region [ 21 , 26 , 27 ] and the length of the gap region of ASOs [ 28 , 29 ].…”

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers

“…We envision that this could be a useful molecular biology tool for induction of RNA cleavages at precisely defined positions. Moreover, when coupled with insights about RNase H sensitivity to base mismatches [ 31 ], it may lead to the development of better SNP selective ASOs.…”