Abstract
Chemical probing methods are crucial to our understanding of the structure and function of RNA molecules. The majority of chemical methods used to probe RNA structure report on Watson–Crick pairing, but tertiary structure parameters such as solvent accessibility can provide an additional layer of structural information, particularly in RNA-protein complexes. Herein we report the development of Light Activated Structural Examination of RNA by high-throughput sequencing, or LASER-Seq, for measuring RNA structure in cells with deep sequencing. LASER relies on a light-generated nicotinoyl nitrenium ion to form covalent adducts with the C8 position of adenosine and guanosine. Reactivity is governed by the accessibility of C8 to the light-generated probe. We compare structure probing by RT-stop and mutational profiling (MaP), demonstrating that LASER can be integrated with both platforms for RNA structure analyses. We find that LASER reactivity correlates with solvent accessibility across the entire ribosome, and that LASER can be used to rapidly survey for ligand binding sites in an unbiased fashion. LASER has a particular advantage in this last application, as it readily modifies paired nucleotides, enabling the identification of binding sites and conformational changes in highly structured RNA.
Highlights
RNA molecules play essential roles in nearly every step of gene regulation, from chromatin modification and transcription to translation regulation
Several existing chemical methods directly measure RNA structure, both inside and outside of living cells. Conventional chemical probes such as dimethyl sulfate (DMS, which methylates the Watson–Crick face of single-stranded adenosine and cytosine residues, as well as the 7 position of guanosine) and SHAPE report primarily on the Watson–Crick pairing status of individual nucleotides [3,4,5]
For reverse transcriptases (RTs) stop analysis, nicotinoyl azide (NAz) reactivity is expressed as Reads Per Million (RPM): the number of reads with 5 ends mapping 1nt 3 of the nucleotide divided by the number of reads mapping to the ribosomal RNA (rRNA)
Summary
RNA molecules play essential roles in nearly every step of gene regulation, from chromatin modification and transcription to translation regulation. Several existing chemical methods directly measure RNA structure, both inside and outside of living cells. Conventional chemical probes such as dimethyl sulfate (DMS, which methylates the Watson–Crick face of single-stranded adenosine and cytosine residues, as well as the 7 position of guanosine) and SHAPE (selective 2 -hydroxyl acylation analyzed by primer extension, which modifies any nucleotide by 2 -hydroxyl acylation at flexible sites) report primarily on the Watson–Crick pairing status of individual nucleotides [3,4,5]. A critical component of the RNA structure toolbox is the ability to interrogate the surface opposite the Watson–Crick face to obtain a more general map of nucleobase solvent accessibility. While HRF is implemented in vitro, in vivo probing requires a synchrotron X-ray source [7]
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