Oligonucleotide drugs, an emerging modulator class, hold promise for targeting previously undruggable biomacromolecules. To date, only 18 oligonucleotide drugs, including sought-after antisense oligonucleotides (ASO) and splice-switching oligonucleotides (SSO), have FDA approval. These agents effectively bind mRNA, inducing degradation or modulating splicing. Current oligonucleotide drug design strategies prioritize full Watson-Crick base pair complementarity, overlooking mRNA target 3D shapes. Given that mRNA conformational diversity can impact hybridization, incorporating mRNA key-structural properties into the design may expedite ASO lead discovery. Using atomistic molecular dynamics simulations inspired by experimental data, we demonstrate the advantages of incorporating common triple base pairs into the design of antisense oligonucleotides (ASOs) targeting RNA hairpin motifs, which are highly accessible regions for interactions. By employing an RNA pseudoknot modified into an ASO-hairpin complex, we investigate the effects of ASO length and hairpin loop mutations. Our findings suggest that ASO-mRNA complex stability is influenced by ASO length, number of common triple base pairs, and the dynamic accessibility of bases in the hairpin loop. Our study offers new mechanistic insights into ASO-mRNA complexation and underscores the value of pseudoknots in constructing training datasets for machine learning models aimed at designing novel ASO leads.

中文翻译：

---

ASO-RNA 复合的机制见解：推进反义寡核苷酸设计策略。

寡核苷酸药物是一种新兴的调节剂类别，有望针对以前无法成药的生物大分子。迄今为止，只有 18 种寡核苷酸药物获得 FDA 批准，包括广受欢迎的反义寡核苷酸 (ASO) 和剪接转换寡核苷酸 (SSO)。这些试剂有效结合 mRNA，诱导降解或调节剪接。目前的寡核苷酸药物设计策略优先考虑完整的 Watson-Crick 碱基对互补性，忽略了 mRNA 目标 3D 形状。鉴于 mRNA 构象多样性会影响杂交，将 mRNA 关键结构特性纳入设计中可能会加快 ASO 先导化合物的发现。利用受实验数据启发的原子分子动力学模拟，我们证明了将常见三碱基对纳入针对 RNA 发夹基序的反义寡核苷酸 (ASO) 设计中的优势，这些基序是非常容易发生相互作用的区域。通过采用修饰为 ASO-发夹复合物的 RNA 假结，我们研究了 ASO 长度和发夹环突变的影响。我们的研究结果表明，ASO-mRNA 复合物的稳定性受到 ASO 长度、常见三碱基对的数量以及发夹环中碱基的动态可及性的影响。我们的研究为 ASO-mRNA 复合提供了新的机制见解，并强调了假结在为旨在设计新型 ASO 线索的机器学习模型构建训练数据集方面的价值。