Laboratory of Frontier Nucleic Acid Technologies in Gene Therapy of Cancer, SCAMT Institute, ITMO University, Saint-Petersburg, 191002, Russia.
Chemistry Department, University of Central Florida, Orlando, FL 32816, USA.
Nucleic Acids Res. 2024 Jun 10;52(10):5866-5879. doi: 10.1093/nar/gkae295.
Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.
多价识别和结合生物分子是一种自然现象，它可以在不降低识别特异性的情况下增加结合稳定性（亲和力）。在这项研究中，我们利用这一现象来提高 DNA 酶（Dz）切割 RNA 的效率并保持其高特异性。我们设计了一系列包含两个 Dz 分子的 DNA 构建体，称为双价 Dz 器件（BDD）。与传统设计的 Dz 相比，一个 BDD 将折叠 RNA 片段的切割效率提高了 17 倍。这种提高是由于两个催化核心提高了 RNA 结合能力和 RNA 切割的概率。通过调节 BDD 中 Dz 分子的缔合程度，我们实现了对单碱基错配 RNA 的优异选择性，同时保持相对较高的切割速率。此外，三价 Dz 在切割折叠 RNA 方面的效率甚至比 BDD 更高。数据表明，几个 RNA 切割单元的协同作用可以显著提高 RNA 切割的效率并保持其高特异性，这对于开发基于 Dz 的基因敲低剂非常重要。
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