کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1175289 1491337 2016 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Optimization of high-throughput sequencing kinetics for determining enzymatic rate constants of thousands of RNA substrates
چکیده انگلیسی

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 investigated 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 found that high-throughput sequencing and experimental reproducibility contribute to error, and these are the main sources of imprecision in the quantified results when otherwise optimized guidelines are followed.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 510, 1 October 2016, Pages 1–10
نویسندگان
, , ,