The discovery of functional RNAs that are critical for normal and disease physiology continues to expand at a breakneck pace. Many RNA functions are controlled by the formation of specific structures, and an understanding of each structural component is necessary to elucidate its function. Measuring solvent accessibility intracellularly with experimental ease is an unmet need in the field. Here, we present a novel method for probing nucleobase solvent accessibility, Light Activated Structural Examination of RNA (LASER). LASER depends on light activation of a small molecule, nicotinoyl azide (NAz), to measure solvent accessibility of purine nucleobases. In vitro, this technique accurately monitors solvent accessibility and identifies rapid structural changes resulting from ligand binding in a metabolite-responsive RNA. LASER probing can further identify cellular RNA–protein interactions and unique intracellular RNA structures. Our photoactivation technique provides an adaptable framework to structurally characterize solvent accessibility of RNA in many environments.
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We thank members of the Spitale lab for their careful reading and critique of the manuscript. The Spitale lab is supported by startup funds from the University of California, Irvine, and the NIH (1DP2GM119164 RCS) and 1RO1MH109588 (RCS). F.F. and the computational work are supported by the US Department of Energy under Award DE-SC0008694. C.M.H. acknowledges financial support from the National Science Foundation (DMR-1212842 and CHE-1609889), as well as generous allocations of computational resources at the Ohio Supercomputer Center. Femtosecond TRIR experiments were performed at The Ohio State University's Center for Chemical and Biophysical Dynamics.
The authors declare no competing financial interests.
Supplementary Tables 1–8, Supplementary Figures 1–16 (PDF 2533 kb)
Cartesian Coordinates of Structures presented in Supplementary Figures 1 and 2. (XLSX 69 kb)
Cartesian Coordinates of Structures presented in Supplementary Figures 3–6. (XLSX 47 kb)
LASER (+SAM) and corresponding solvent accessibility measurements at C8. Values are in A2. Solvent was contoured to 2 Å. (XLSX 44 kb)
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Feng, C., Chan, D., Joseph, J. et al. Light-activated chemical probing of nucleobase solvent accessibility inside cells. Nat Chem Biol 14, 276–283 (2018). https://doi.org/10.1038/nchembio.2548
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