Visualizing RNA Conformational Changes via Pattern Recognition of RNA by Small Molecules

Abstract

Conformational changes in RNA play vital roles in the regulation of many biological systems, yet these changes can be challenging to visualize. Previously, we demonstrated that Pattern Recognition of RNA by Small Molecules (PRRSM) can unbiasedly cluster defined RNA secondary structure motifs utilizing an aminoglycoside receptor library. In this work, we demonstrate the power of this method to visualize changes in folding at the secondary structure level within two distinct riboswitch structures. After labeling at three independent positions on each riboswitch, PRRSM accurately classified all apo and ligand-bound riboswitch structures, including changes in the size of a structural motif, and revealed modification sites that prevented folding and/or led to a mixture of states. These data underscore the utility and robustness of the PRRSM assay for rapid assessment of RNA structural changes and for gaining ready insight into nucleotide positions critical to RNA folding.

A transient conformation facilitates ligand binding to the adenine riboswitch

“…Detecting transient conformations is pivotal for uncovering dynamic biological processes and important in designing novel strategies for controlling riboswitches. For example, characterizing transient conformations can aid in the development of new antibiotics that selectively target transient conformations ( Eubanks et al., 2019 ; Jones and Ferré-D’Amaré, 2017 ; Lund et al., 2020 ; Rode et al., 2018 ). No transient conformations of the full-length adenine riboswitch have been reported.…”

Machine Learning Aids Classification and Discrimination of Noncanonical DNA Folding Motifs by an Arrayed Host:Guest Sensing System

“…Pattern-recognition-based differential sensing can be a powerful tool for creating fluorescent probes that selectively recognize and differentiate DNA folding. This has been used to identify folding patterns in fluorescently labeled RNAs, and fluorescence displacement assays paired with multivariate analysis allow classification of DNA structure or identify ligands that can bind these structures …”

RNA Structural Differentiation: Opportunities with Pattern Recognition

“…In addition to providing a better understanding of RNA molecular recognition, we evaluated whether the classification power of PRRSM could generate insight into site-specific RNA structure. We analyzed biologically relevant RNA constructs of known structure with multiple secondary structure motifs and/or inducible conformational changes . We fluorescently labeled each construct with BFU at nucleotide positions of interest.…”

“…PCA plot of PreQ1 U9 and FR U9 BFU constructs with and without a ligand. Also, U25 and G33 TAR BFU constructs cluster for bulge and hairpin secondary structures, respectively …”

“…We analyzed biologically relevant RNA constructs of known structure with multiple secondary structure motifs and/ or inducible conformational changes. 221 We fluorescently labeled each construct with BFU at nucleotide positions of interest. To begin, we examined a truncated version of the HIV-1 TAR RNA consisting of a three-nucleotide bulge and a six-nucleotide hairpin.…”