Institute of Biochemistry and Biotechnology, Section Microbial Biotechnology, Martin Luther University Halle-Wittenberg, D-06120 Halle (Saale), Germany.
Institute for Sustainable Plant Protection, Department of Biology, Agricultural and Food Sciences National Research Council, Bari Unit, I-70126 Bari, Italy.
Int J Mol Sci. 2023 Dec 5;24(24):17153. doi: 10.3390/ijms242417153.
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.
反义寡核苷酸 (ASO) 长期以来一直被用于选择性地抑制或调节 RNA 水平的基因表达，一些 ASO 已被批准用于临床使用。然而，反义技术的实用性仍然受到限制，因为难以可靠地预测复杂折叠 RNA 中 ASO 可接近的位点。最近，我们应用了一种在体外重现 RNA 诱导的 RNA 沉默的植物方法，可靠地鉴定了靶 RNA 中可被小干扰 RNA(siRNA)引导的 Argonaute 内切酶接近的位点。在这里，我们表明该方法也适用于鉴定通过 RNase H 诱导的 DNA 诱导的 RNA 沉默有效的 ASO。我们表明，针对病毒基因组的以这种方式鉴定的 ASO 在保护植物免受感染方面与具有相应序列的 siRNA 同样有效。通过化学修饰可以进一步增强 ASO 的抗病毒活性。这得出了两个重要结论：可以使用相同的方法鉴定能够有效敲低复杂 RNA 分子的 siRNA 和 ASO，并且以这种方式优化的 ASO 可以在作物保护中找到应用。这里开发的技术不仅可用于植物中有效的 RNA 沉默，也可用于其他生物体。
