RNA base pairing complexity in living cells visualized by correlated chemical probing

Abstract

2 ABSTRACT RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells. !

To knot or not to knot: Multiple conformations of the SARS-CoV-2 frameshifting RNA element

“…79 The resulting parsed mutation files were used in the Pair-MaP pipeline which uses PairMapper and RingMapper to compute and identify correlated mutations in the DMS-MaP sequencing dataset. 57 The correlated mutational outputs were plotted with arcPlot. 57…”

Functional and structural basis of extreme non-coding conservation in vertebrate 5’UTRs

“…We chose to mask the nucleotide positions which average count less than the threshold. This results in filtering of nearly all G nucleotides as expected, which have lower signal (lower modification-mutation rates) than other bases and different variance distribution 70 . The diagonal of the matrix within 4nt is also masked due to extremely low co-variation.…”

“…While previous genome-wide in vivo chemical probing experiments provided key global insights, they were limited in their statistical resolution on many mRNA regions due to the lack of high coverage necessary to resolve localized accessibility pattern difference between different conditions 63–65 . To obtain a high coverage accessibility data for a large number of h5UTRs, we performed a highly multiplexed adaptation of dimethyl sulfate (DMS) mutational profiling 68–70 . In DMS mutational profiling, adduct-induced reverse transcriptase misincorporation of dNTPs into cDNAs are used as the readout of DMS modification rates.…”

“…2.5mL ice-cold 30% BME (MilliporeSigma, M3148) in ethanol is added to quench the reaction. This DMS modification protocol is adapted from protocol and data reported in 70 . Following centrifuge to remove the supernatant, the cells are lysed in Trizol (ThermoFisher Scientific, 15596026).…”

Measuring thermodynamic preferences to form non-native conformations in nucleic acids using melting experiments reveals a rich sequence-specific DNA conformational landscape

“…This is because measuring the energetic cost to form non-native conformations requires accurately determining their low abundance in a sea of other conformations in the apo-ensemble. A variety of approaches have been used to measure such conformational equilibria, including Nuclear Magnetic Resonance (NMR) (33), Electron Paramagnetic Resonance (EPR) (34) and Infrared (IR) spectroscopy (35), Fluorescence Resonance Energy Transfer (FRET) (36), Cryo-EM and X-ray crystallography (37), and chemical probing (38). However, with the exception of NMR, most of these techniques are not applicable to conformations such as Hoogsteen bps and the base open states, which have populations <1%, are short-lived with lifetimes shorter than a few milliseconds, and which involve localized bp rearrangements.…”