Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA

Abstract

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms.

Potent and selective ‘genetic zipper’ method for plant protection: innovative DNA psyllidicides againstTrioza alacrisFlor based on short unmodified antisense oligonucleotides targeting rRNA of the pest

“…In addition to better target specificity, antisense oligonucleotides are easier and cheaper to synthesize than siRNAs or dsRNAs, and in the human system, antisense oligonucleotides have been shown to have low immunoreactivity, which is also of significant importance for potential applications in edible plants that could accumulate this kind of means of insect pest control. It is important to note, from the point of view of environmental risks, there are no findings in the numerous human clinical studies that prove, for example, genomic integration events attributable to the use of antisense oligonucleotides (Gruber et al 2023).…”