Oberemok Vol, Gal'chinsky Nikita, Novikov Ilya, Sharmagiy Alexander, Yatskova Ekaterina, Laikova Ekaterina, Plugatar Yuri
Department of General Biology and Genetics, Institute of Biochemical Technologies, Ecology and Pharmacy, V.I. Vernadsky Crimean Federal University, Simferopol 295007, Russia.
Laboratory of Entomology and Phytopathology, Dendrology and Landscape Architecture, Nikita Botanical Gardens-National Scientific Centre of the Russian Academy of Sciences, Yalta 298648, Russia.
Int J Mol Sci. 2025 Aug 4;26(15):7530. doi: 10.3390/ijms26157530.
Contact unmodified antisense DNA biotechnology (CUADb), developed in 2008, employs short antisense DNA oligonucleotides (oligos) as a novel approach to insect pest control. These oligonucleotide-based insecticides target pest mature rRNAs and/or pre-rRNAs and have demonstrated high insecticidal efficacy, particularly against sap-feeding insect pests, which are key vectors of plant DNA viruses and among the most economically damaging herbivorous insects. To further explore the potential of CUADb, this study evaluated the insecticidal efficacy of short 11-mer antisense DNA oligos against , in comparison with long 56-mer single-stranded and double-stranded DNA sequences. The short oligos exhibited higher insecticidal activity. By day 9, the highest mortality rate (97.66 ± 4.04%) was recorded in the Coccus-11 group, while the most effective long sequence was the double-stranded DNA in the dsCoccus-56 group (77.09 ± 6.24%). This study also describes the architecture of the DNA containment (DNAc) mechanism, highlighting the intricate interactions between rRNAs and various types of DNA oligos. During DNAc, the Coccus-11 treatment induced enhanced ribosome biogenesis and ATP production through a metabolic shift from carbohydrates to lipid-based energy synthesis. However, this ultimately led to a 'kinase disaster' due to widespread kinase downregulation resulting from insufficient ATP levels. All DNA oligos with high or moderate complementarity to target rRNA initiated hypercompensation, but subsequent substantial rRNA degradation and insect mortality occurred only when the oligo sequence perfectly matched the rRNA. Both short and long oligonucleotide insecticide treatments led to a 3.75-4.25-fold decrease in rRNA levels following hypercompensation, which was likely mediated by a DNA-guided rRNase, such as RNase H1, while crucial enzymes of RNAi (DICER1, Argonaute 2, and DROSHA) were downregulated, indicating fundamental difference in molecular mechanisms of DNAc and RNAi. Consistently, significant upregulation of RNase H1 was detected in the Coccus-11 treatment group. In contrast, treatment with random DNA oligos resulted in only a 2-3-fold rRNA decrease, consistent with the normal rRNA half-life maintained by general ribonucleases. These findings reveal a fundamental new mechanism of rRNA regulation via complementary binding between exogenous unmodified antisense DNA and cellular rRNA. From a practical perspective, this minimalist approach, applying short antisense DNA dissolved in water, offers an effective, eco-friendly and innovative solution for managing sternorrhynchans and other insect pests. The results introduce a promising new concept in crop protection: DNA-programmable insect pest control.
接触未修饰反义DNA生物技术(CUADb)于2008年开发,采用短反义DNA寡核苷酸(oligos)作为控制害虫的新方法。这些基于寡核苷酸的杀虫剂靶向害虫成熟rRNA和/或前体rRNA,并已证明具有高杀虫效果,特别是针对吸食汁液的害虫,这些害虫是植物DNA病毒的关键传播媒介,也是最具经济破坏力的食草昆虫之一。为了进一步探索CUADb的潜力,本研究评估了短11聚体反义DNA寡核苷酸与长56聚体单链和双链DNA序列相比对[具体害虫名称缺失]的杀虫效果。短寡核苷酸表现出更高的杀虫活性。到第9天,Coccus - 11组记录到最高死亡率(97.66 ± 4.04%),而最有效的长序列是dsCoccus - 56组中的双链DNA(77.09 ± 6.24%)。本研究还描述了DNA遏制(DNAc)机制的结构,强调了rRNA与各种类型DNA寡核苷酸之间复杂的相互作用。在DNAc过程中,Coccus - 11处理通过从碳水化合物到基于脂质的能量合成的代谢转变诱导核糖体生物合成增强和ATP产生。然而,由于ATP水平不足导致广泛的激酶下调,这最终导致了“激酶灾难”。所有与靶标rRNA具有高或中度互补性的DNA寡核苷酸都引发了超补偿,但只有当寡核苷酸序列与rRNA完美匹配时,随后才会发生大量rRNA降解和昆虫死亡。短和长寡核苷酸杀虫剂处理在超补偿后均导致rRNA水平下降3.75 - 4.25倍,这可能由DNA引导的rRNase(如RNase H1)介导,而RNAi的关键酶(DICER1、Argonaute 2和DROSHA)被下调,表明DNAc和RNAi在分子机制上存在根本差异。一致地,在Coccus - 11处理组中检测到RNase H1的显著上调。相比之下,用随机DNA寡核苷酸处理仅导致rRNA下降2 - 3倍,这与一般核糖核酸酶维持的正常rRNA半衰期一致。这些发现揭示了通过外源未修饰反义DNA与细胞rRNA之间的互补结合进行rRNA调控的全新机制。从实际角度来看,这种将短反义DNA溶解在水中的极简方法为管理粉虱类害虫和其他害虫提供了一种有效、环保且创新的解决方案。这些结果在作物保护方面引入了一个有前景的新概念:DNA可编程害虫控制。
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