The influence of antisense oligonucleotide-induced RNA structure on Escherichia coli RNase H1 activity.

Walt F Lima,Venkatraman Mohan,Stanley T Crooke

doi:10.1074/jbc.272.29.18191

Walt F Lima, Venkatraman Mohan + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.29.18191

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Abstract

The ability of Escherichia coli RNase H1 to hydrolyze structured substrates containing antisense oligonucleotides preannealed to a 47-mer RNA was compared with its ability to hydrolyze unstructured substrates containing antisense oligonucleotides duplexed with 13-mer RNA. These results demonstrate that when antisense oligonucleotides were bound to structured RNA, the resultant duplexes were cleaved at rates significantly slower than when the same oligonucleotides were bound to unstructured oligoribonucleotides. Structured substrates exhibited fewer cleavage sites, and each cleavage site was cleaved less rapidly than in unstructured substrates. Furthermore, the enzymatic activity of E. coli RNase H1 for the structured substrates was most affected when the cleavage sites corresponding to the enzymatically most active sites on the unstructured substrates were blocked in the structured substrates. Molecular modeling suggests that the observed ablation of RNase H activity was due to the steric hindrance of the enzyme by the structured RNA, i.e. steric interference of the phosphate groups on the substrate and/or the binding site of the enzyme. When chimeric oligonucleotides composed of a five-base deoxynucleotide sequence flanked by chemically modified nucleotides were bound to structured RNA, the resultant duplexes were even worse substrates for RNase H. These results offer further insights into the role of antisense-induced RNA structure on RNase H activity and may facilitate the design of effective antisense oligonucleotides.

Highlights

One mechanism of action commonly employed by antisense oligonucleotides is the induction of cellular enzymes such as RNase H to degrade the target RNA [7,8,9]
The resulting antisense oligonucleotide-induced 47mer RNA structures consist of a quasi-continuous helix formed by the double-strand RNA stem and the antisense oligonucleotide annealed to the 47-mer RNA
These antisense oligonucleotide-induced 47-mer RNA structures are divided into two categories: loop 1 (L1) pseudo-half-knots formed by antisense oligonucleotides targeting the 39 side of the loop region on the parent 47-mer RNA hairpin (e.g. 26 –9, 24 –11, and 22–13), and loop 2 (L2) pseudo-half-knots formed by oligonucleotides targeting the 59 side of the loop region on the parent 47-mer RNA hairpin (e.g. 16 –9, 16 –11, and 16 –13)

Summary

Introduction

One mechanism of action commonly employed by antisense oligonucleotides is the induction of cellular enzymes such as RNase H to degrade the target RNA [7,8,9]. The protection of the antisense oligonucleotide binding sites on the 47-mer RNA from single-strand-specific ribonuclease digestion suggests that, with the exception of the 16 –13 pseudo-half-knot, the oligonucleotides were completely base paired with the 47-mer RNA (Fig. 1A).

Results

Conclusion