Chimeric Flaviviral RNA-siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells.

Cressida Harvey,Jonathan Hall,Alyssa C Hill,Sven Klassa,Esteban Finol

doi:10.1002/cbic.202100434

Abstract

RNA is an emerging platform for drug delivery, but the susceptibility of RNA to nuclease degradation remains a major barrier to its implementation in vivo. Here, we engineered flaviviral Xrn1‐resistant RNA (xrRNA) motifs to host small interfering RNA (siRNA) duplexes. The xrRNA‐siRNA molecules self‐assemble in vitro, resist degradation by the conserved eukaryotic 5’ to 3’ exoribonuclease Xrn1, and trigger gene silencing in 293T cells. The resistance of the molecules to Xrn1 does not translate to stability in blood serum. Nevertheless, our results demonstrate that flavivirus‐derived xrRNA motifs can confer Xrn1 resistance on a model therapeutic payload and set the stage for further investigations into using the motifs as building blocks in RNA nanotechnology.

Highlights

Ribonucleic acid (RNA) is an emerging platform for drug delivery.[1]
The 5’-phosphorylated 21mers were degraded within 20 min, while the 5’-phosphorylated Xrn1-resistant RNA (xrRNA) remained intact over this period (Figure 1c). These results indicate that the designed xrRNA constructs adopt their native, Xrn1-resistant 3D fold and further confirm that the stemloop P4À L4 is not required for Xrn1 resistance
The molecules triggered silencing of a Renilla luciferase reporter in 293T cells with a potency comparable to a naked small interfering RNA (siRNA) molecule

Summary

Introduction

Ribonucleic acid (RNA) is an emerging platform for drug delivery.[1] RNA is biocompatible and exhibits chemical, structural, and functional modularity.[2] motifs derived from biological or synthetic RNA molecules can be combined into single, multifunctional RNA structures.[3] Branched RNA molecules,[4] RNA nano-cubes,[5] and RNA nano-rings[5b,6] have all shown promise as vehicles for the delivery of small interfering RNA (siRNA) duplexes Their advantages include one-pot selfassembly,[4,5,6] tissue-specific delivery,[4a–e,g] enhanced cellular uptake,[4g–j,5b,6b] alternative processing by the RNA interference (RNAi) machinery,[4f,h,j] more potent or prolonged RNAi activity,[4i–k,5b] and synergistic or combinatorial RNAi.[4f–k,5a,6b] the susceptibility of RNA to nuclease degradation is still a major barrier to its development for applications in vivo. This work demonstrates that xrRNA motifs can protect a model therapeutic payload from Xrn1-mediated decay and lays the groundwork for continued investigations into using the motifs as building blocks in RNA nanotechnology

Results and Discussion

Conclusion

Conflict of Interest