Kinetics and equilibrium constants of oligonucleotides at low concentrations. Hybridization and melting study
Kinetics and equilibrium constants of oligonucleotides at low concentrations. Hybridization and melting study
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DOI：
10.1039/c9cp01295h
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发表时间：
2019-05-28
影响因子：
3.3
通讯作者：
Holyst, Robert
中科院分区：
文献类型：
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作者：
Bielec, Krzysztof;Sozanski, Krzysztof;Holyst, Robert
虽然DNA杂交/融化是最重要的生化反应之一，但该过程的非平凡动力学还没有完全被理解。在这项工作中，我们使用Forster共振能量转移(FRET)研究了温度、离子强度和寡核苷酸长度对DNA寡核苷酸结合和解离的动力学常数和平衡常数的影响。我们表明，在低试剂浓度和离子强度下，建立单链和双链形式之间的平衡所需的时间可能是几天的量级，即使对于长度为20个碱基对或更少的简单寡核苷酸也是如此。我们还识别和讨论了在极稀溶液中进行的基于荧光的实验可能存在的伪影。这些结果应该被证明对基于DNA匹配的技术的明智设计是有用的，包括传感器、DNA增殖、测序和基因操作。
Although DNA hybridization/melting is one of the most important biochemical reactions, the non-trivial kinetics of the process is not yet fully understood. In this work, we use Forster resonance energy transfer (FRET) to investigate the influence of temperature, ionic strength, and oligonucleotide length on the kinetic and equilibrium constants of DNA oligonucleotide binding and dissociation. We show that at low reagent concentrations and ionic strength, the time needed to establish equilibrium between single and double strand forms may be of the order of days, even for simple oligonucleotides of a length of 20 base pairs or less. We also identify and discuss the possible artifacts related to fluorescence-based experiments conducted in extremely dilute solutions. The results should prove useful for the judicious design of technologies based on DNA-matching, including sensors, DNA multiplication, sequencing, and gene manipulation.