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- W4220989096 abstract "RNA Bind-n-Seq (RBNS) is a cost-effective, high-throughput method capable of identifying the sequence preferences of RNA-binding proteins and of qualitatively defining relative dissociation constants. Although RBNS is often described as an unbiased method, several factors may influence the outcome of the analysis. Here, we discuss these biases and present an analytical strategy to estimate absolute binding affinities from RBNS data, extend RBNS to kinetic studies, and develop a framework to compute relative association and dissociation rate constants. As proof of principle, we measured the equilibrium binding properties of mammalian Argonaute2 (AGO2) guided by eight microRNAs (miRNAs) and kinetic parameters for let-7a. The miRNA-binding site repertoires, dissociation constants, and kinetic parameters calculated from RBNS data using our methods correlate well with values measured by traditional ensemble and single-molecule approaches. Our data provide additional quantitative measurements for Argonaute-bound miRNA binding that should facilitate development of quantitative targeting rules for individual miRNAs." @default.
- W4220989096 created "2022-04-03" @default.
- W4220989096 creator A5020225652 @default.
- W4220989096 creator A5031216320 @default.
- W4220989096 creator A5059718607 @default.
- W4220989096 date "2022-03-01" @default.
- W4220989096 modified "2023-10-09" @default.
- W4220989096 title "Principles and pitfalls of high-throughput analysis of microRNA-binding thermodynamics and kinetics by RNA Bind-n-Seq" @default.
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- W4220989096 doi "https://doi.org/10.1016/j.crmeth.2022.100185" @default.
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- W4220989096 hasPublicationYear "2022" @default.
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