Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence
Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence
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DOI：
10.1093/nar/gkp884
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发表时间：
2010-01-01
影响因子：
14.9
通讯作者：
Raghunath, Michael
中科院分区：
文献类型：
--
作者：
Harve, Karthik S.;Lareu, Ricky;Raghunath, Michael
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，需要热循环来解链DNA链、引物-模板杂交和酶促引物延伸。引物退火和延伸的最适温度是惊人不同的，这预示着引物在延伸期间与模板解离，从而损害PCR效率。我们假设MMC不仅对体内的延伸阶段很重要，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，MMC稳定互补核苷酸之间的氢键。MMC下的实时PCR证实，互补DNA-DNA和DNA-RNA杂交体的解链温度提高了8摄氏度，具有高特异性和延伸后的高保留性（71%对37%非拥挤）。MMC增强DNA杂交螺旋性，并驱动双链体形成的特异性，偏好匹配与错配序列，包括发夹形成DNA单链。
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.