Post-SELEX modifications with locked nucleic acids (LNA) of a SEA-specific DNA aptamer assisted by in silico modelling†

IF 3.2 3区 工程技术 Q2 CHEMISTRY, PHYSICAL Molecular Systems Design & Engineering Pub Date : 2024-05-28 DOI:10.1039/D4ME00043A
Ricardo Oliveira, Eva Pinho, Nuno Filipe Azevedo and Carina Almeida
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Abstract

Post-SELEX modifications assisted by in silico modelling are powerful tools to improve the performance of aptamers, by providing a rational approach for the selection of modified-versions of aptamers. In this study, a complete in silico analysis of the three-dimensional structure of a previously selected DNA aptamer (Apt5) against staphylococcal enterotoxin A (SEA) was performed. Locked nucleic acid (LNA) modifications were introduced in key locations and their effect on the aptamer structure and docking were evaluated. Promising LNA aptamers were then synthetized and their dissociation constants (KD), as well as stability, were evaluated. From the in silico analysis, it was possible to identify three promising LNA variations that did not affect drastically the three-dimensional structure and the molecular docking with the toxin. The KD of the LNA aptamers were higher than the DNA aptamer (Apt5: KD = 13 ± 2 nM, LNA13: KD = 157 ± 39 nM, LNA14: KD = 74 ± 24 nM, LNA15: KD = 143 ± 28 nM), but remained in the low nanomolar range. Even so, the KD of LNA14 was not significantly different (P < 0.05) compared to the value of the original aptamer and the introduction of LNA increased its thermal stability, increasing the range of functionality of the original aptamer. However, the introduced modifications were not enough to increase the biological stability of the aptamer, remaining susceptible to a complete degradation by endonucleases and exonucleases in 5 minutes. Altough partial modifications with LNA may not be able to overcome all the limitations of DNA aptamers, post-SELEX modifications assisted by in silico modelling have shown promising results in predicting functional modified aptamers, avoiding a time-consuming and expensive trial and error approach.

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用锁定核酸 (LNA) 对一种 SEA 特异性 DNA 类似物进行 SELEX 后修饰,并辅以硅学建模
通过硅学建模辅助 SELEX 后修饰是提高适配体性能的有力工具,它为选择修饰后的适配体提供了一种合理的方法。在本研究中,我们对之前筛选出的针对葡萄球菌肠毒素 A(SEA)的 DNA 短肽(Apt5)的三维结构进行了完整的硅学分析。在关键位置引入了锁定核酸(LNA)修饰,并评估了它们对适配体结构和对接的影响。然后合成了有前景的 LNA 合酶,并评估了它们的解离常数(KD)和稳定性。通过硅学分析,确定了三种有前景的 LNA 变体,它们不会对三维结构和与毒素的分子对接产生重大影响。LNA 嵌合体的 KD 值高于 DNA 嵌合体(Apt5:KD = 13 ± 2 nM;LNA13:KD = 157 ± 39 nM;LNA14:KD = 74 ± 24 nM;LNA15:KD = 143 ± 28 nM),但仍保持在较低的纳摩尔范围内。即便如此,LNA14 的 KD 与原始适配体的 KD 值相比并无显著差异(P < 0.05),而且 LNA 的引入增加了其热稳定性,扩大了原始适配体的功能范围。然而,引入的修饰并不足以提高链 接酶的生物稳定性,它仍然容易在 5 分钟内被内切酶和外切酶完全降解。虽然用 LNA 进行部分修饰可能无法克服 DNA 类似物的所有局限性,但在硅学建模的辅助下,SELEX 后修饰在预测功能性修饰类似物方面显示出了很好的效果,避免了耗时且昂贵的反复试验方法。
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来源期刊
Molecular Systems Design & Engineering
Molecular Systems Design & Engineering Engineering-Biomedical Engineering
CiteScore
6.40
自引率
2.80%
发文量
144
期刊介绍: Molecular Systems Design & Engineering provides a hub for cutting-edge research into how understanding of molecular properties, behaviour and interactions can be used to design and assemble better materials, systems, and processes to achieve specific functions. These may have applications of technological significance and help address global challenges.
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