RNA-binding proteins (RBPs) are abundant across species and perform diverse critical roles, ranging from RNA nuclear export to viral genomic RNA packaging. Understanding the affinity of the RBPs for their RNA targets in terms of the dissociation constant (K d ), offers insights into the mechanism of binding as well as the overall biological significance of the interaction, potentially identifying targets for therapeutic intervention. Existing methods for measuring the binding affinity either lack physiological context or are limited by intrinsic biases introduced during the assays. Currently, quantitative RNA-protein binding affinities can only be measured in vitro, and methods to identify RNA-protein binding partners in the cellular context remain qualitative. Our solution utilizes the trans-activating regulatory protein (Tat) of HIV-1 that binds to an RNA stem loop structure found in the HIV-I 5’ UTR known as the transactivation response element (TAR). By modifying this RBP-RNA pair, we aim to develop a general assay that can measure K d values of protein-RNA interactions in vivo. This system has several unique properties that make it ideal for this purpose, primarily that the RNA-protein binding event is the rate-limiting step of the biological process that produces our readout, and that the identity of the RNA does not affect subsequent steps. We have applied this assay to three canonical protein-RNA systems (MS2 bacteriophage coat protein-stem loop, Protein N λ -BoxB, and HIV Rev-RRE) and have also made preliminary optimizations to streamline the protocol and improve the signal-to-noise ratio. Moving forward, we would test the sensitivity of the assay using mutant RBPs and RNA sequences.
Guo et al. (Sun,) studied this question.