Tsai Yi-Chen, Chen Wen-Yih, Chiu Chi-Cheng
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
Department of Chemical and Materials Engineering, National Central University, Taoyuan 32001, Taiwan.
Comput Struct Biotechnol J. 2023 Feb 24;21:1820-1827. doi: 10.1016/j.csbj.2023.02.043. eCollection 2023.
Polymerase chain reaction (PCR) is a powerful molecular biology assay for gene detection and quantification. Conventional DNA primers for PCR often suffer from poor sensitivity in specific gene detection. Recently, oligonucleotides containing methyl phosphotriester (MPTE-DNA) have been developed with enhanced DNA hybridization and improved gene detection sensitivity. Yet, site-specific MPTE-modifications on DNA primers have been reported to affect PCR amplification efficiencies while the detailed mechanism remains elusive. Here, we utilized molecular dynamics (MD) simulation to examine the effects of site-specific MPTE-modified primers on the structure and motions of DNA/Taq polymerase complexes. All tested MPTE-DNA/Taq complexes exhibited RMSD values below 2 Å, indicating insignificant effects of all methylation sites on the complex stability. The energetic and hydrogen-bonding analyses suggest minor effects of methylation at t-3, t-4, t-6, and t-7 positions on the DNA-Taq interaction. Principal component analyses further reveal that only t-3, and t-7 methylations preserve the motions of the Taq thumb domain. The site-specific methylation affects the interaction between DNA and the surrounding protein residues, resulting in allosteric-like effects on the DNA/Taq complex. The MD data complement the best experimentally observed PCR efficacies for the t-3 and t-7 positions among all tested MPTE-primers. The unveiled molecular insights can benefit the design of novel PCR primers for improving genetic testing platforms.
聚合酶链反应（PCR）是一种用于基因检测和定量的强大分子生物学检测方法。用于PCR的传统DNA引物在特定基因检测中往往灵敏度较差。最近，含有甲基磷酸三酯（MPTE-DNA）的寡核苷酸已被开发出来，其具有增强的DNA杂交能力和提高的基因检测灵敏度。然而，据报道，DNA引物上的位点特异性MPTE修饰会影响PCR扩增效率，而详细机制仍不清楚。在这里，我们利用分子动力学（MD）模拟来研究位点特异性MPTE修饰引物对DNA/Taq聚合酶复合物结构和运动的影响。所有测试的MPTE-DNA/Taq复合物的均方根偏差（RMSD）值均低于2 Å，表明所有甲基化位点对复合物稳定性的影响不显著。能量和氢键分析表明，t-3、t-4、t-6和t-7位点的甲基化对DNA-Taq相互作用的影响较小。主成分分析进一步揭示，只有t-3和t-7甲基化保留了Taq拇指结构域的运动。位点特异性甲基化影响DNA与周围蛋白质残基之间的相互作用，从而对DNA/Taq复合物产生类似变构的效应。MD数据补充了所有测试的MPTE引物中t-3和t-7位点最佳的实验观察到的PCR效率。所揭示的分子见解有助于设计新型PCR引物，以改进基因检测平台。
