Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore.
Nucleic Acids Res. 2010 Jan;38(1):172-81. doi: 10.1093/nar/gkp884. Epub 2009 Oct 23.
Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA-DNA and DNA-RNA hybrids increased by up to 8 degrees C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.
体内 DNA 扩增发生在以大分子拥挤（MMC）为特征的细胞内环境中。然而，体外聚合酶链式反应（PCR）是非拥挤的，需要热循环以使 DNA 链熔化、引物-模板杂交和酶促引物延伸。引物退火和延伸的最佳温度差异很大，这预示着引物在延伸过程中会从模板上解离，从而降低 PCR 效率。我们假设 MMC 不仅对体内延伸阶段很重要，而且在 PCR 过程中通过稳定核苷酸杂交体也很重要。新的原子分子动力学模拟阐明了 MMC 稳定了互补核苷酸之间的氢键。在 MMC 下进行的实时 PCR 证实，互补 DNA-DNA 和 DNA-RNA 杂交体的熔化温度在延伸后最多增加了 8°C，具有很高的特异性和双链体保留率（71%比非拥挤时的 37%）。MMC 增强了 DNA 杂交螺旋性，并促使双链体形成特异性，优先选择匹配序列而不是不匹配序列，包括发夹形成的 DNA 单链。
