Abstract
Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.
Highlights
Molecular associations lie at the heart of biology
We evaluated published binding measurements using RNA-protein interactions as an illustrative example
We surveyed 100 studies that reported equilibrium dissociation constants (KD values) and scored them based on two key criteria for reliable binding measurements: sufficient time to equilibration and proper concentration regime (Figure 1)
Summary
Molecular associations lie at the heart of biology Their thermodynamics provides information critical for deriving a fundamental understanding of molecular functions. Buenrostro et al, 2014; Tome et al, 2014; Lambert et al, 2014; Nutiu et al, 2011; Maerkl and Quake, 2007; Adams et al, 2016; Jain et al, 2017). Given these potentially transformative advances, it is especially timely to assess the accuracy of equilibrium binding measurements. We wanted to know whether current practices are sufficient to ensure reliable and accurate measurements, and whether the reliability of these measurements can be readily ascertained from the information provided in published work
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