Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates.
Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates.
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DOI：
10.1016/j.ab.2016.06.004
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发表时间：
2016-10-01
影响因子：
2.9
通讯作者：
Harris ME
中科院分区：
文献类型：
--
作者：
Niland CN;Jankowsky E;Harris ME
RNA结合蛋白和RNA加工酶的特异性的定量对于理解它们在生物过程中的基本作用是必不可少的。高通量测序动力学（HTS-Kin）使用高通量测序和内部竞争动力学来同时监测RNA加工酶对数千种底物的加工速率常数。这项技术提供了前所未有的洞察底物特异性的tRNA加工核酸内切酶核糖核酸酶P.在这里，我们调查的准确性和鲁棒性的测量与HTS-Kin程序的每一步。我们研究了底物浓度对所观察到的速率常数的影响，确定了最佳的动力学参数，并提供了减少底物群体扩增误差的指导方针。重要的是，我们发现高通量测序和实验再现性贡献了它们自己的误差来源，并且当遵循其他优化的指导方针时，这些是定量结果中不精确的主要来源。
Quantification of the specificity of RNA binding proteins and RNA processing enzymes is essential to understanding their fundamental roles in biological processes. High Throughput Sequencing Kinetics (HTS-Kin) uses high throughput sequencing and internal competition kinetics to simultaneously monitor the processing rate constants of thousands of substrates by RNA processing enzymes. This technique has provided unprecedented insight into the substrate specificity of the tRNA processing endonuclease ribonuclease P. Here, we investigate the accuracy and robustness of measurements associated with each step of the HTS-Kin procedure. We examine the effect of substrate concentration on the observed rate constant, determine the optimal kinetic parameters, and provide guidelines for reducing error in amplification of the substrate population. Importantly, we find that high-throughput sequencing, and experimental reproducibility contribute their own sources of error, and these are the main sources of imprecision in the quantified results when otherwise optimized guidelines are followed.