The continuing discoveries of non-coding RNAs (ncRNAs) and their critical roles in cellular and viral machinery are inspiring novel antibacterial antitumor, and antiviral therapies based on disabling or manipulating the RNAs involved. Unfortunately, our poor biophysical understanding of "how RNAs work" hinders the development of these potentially life-saving efforts. A critical bottleneck has been the inapplicability of crystallography, NMR, phylogenetic analysis, and current chemical methods to determine the partly ordered 3D conformations of non-coding RNAs in all their functional states. To resolve this bottleneck, we have recently invented and benchmarked a two-dimensional "mutate-and-map" (M2) technology. This strategy rapidly and comprehensively determines how every single mutation of an RNA perturbs the 2'-hydroxyl chemical accessibility of every other nucleotide, giving rich information on RNA secondary and tertiary structure.
We aim here to more precisely reveal both canonical base pairs and pervasive A-minor tertiary interactions by coupling M2 to two additional chemistries, flavin-mononucleotide-induced photo-oxidation (M2-FMN) and dimethyl-sulfate alkylation (M2-DMS). We propose a high-throughput M2-rescue approach to validate the resulting inferences through "surgical" double-mutant/rescue experiments. Finally, we will apply these technologies to determine structures of mysterious states and regions in two paradigmatic systems in RNA biophysics, the add adenine-binding riboswitch and the FN double-glycine riboswitch;this critical information is not obtainable with any other approach. We will evaluate success through benchmarks on six ncRNA domains of known structure;through M2-rescue validation;and through adoption of our methods and software tools by the broader biological community. In the same way that 2D spectroscopy transformed NMR approaches to small biomolecule structure, we propose that 2D mutate-and-map technology will transform our understanding of structure in long non-coding RNAs, full-length RNA messages, and entire retroviral genomes targeted for biomedical activation or disruption.

Public Health Relevance

RNA molecules play fundamental roles in transmitting and regulating genetic information in all living systems, including disease-causing bacteria, retroviruses like HIV, and tumor cells. New potentially life-saving therapies that target these RNAs are being hindered by the slow rate of determining RNA folds and conformational changes. Our work aims to resolve this critical bottleneck by advancing a new chemical/computational paradigm for high-throughput RNA structure determination.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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Stanford University
Schools of Medicine
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Cordero, Pablo; Kladwang, Wipapat; VanLang, Christopher C et al. (2014) The mutate-and-map protocol for inferring base pairs in structured RNA. Methods Mol Biol 1086:53-77
Kladwang, Wipapat; Mann, Thomas H; Becka, Alex et al. (2014) Standardization of RNA chemical mapping experiments. Biochemistry 53:3063-5
Tian, Siqi; Cordero, Pablo; Kladwang, Wipapat et al. (2014) High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states. RNA 20:1815-26
Kim, Hanjoo; Cordero, Pablo; Das, Rhiju et al. (2013) HiTRACE-Web: an online tool for robust analysis of high-throughput capillary electrophoresis. Nucleic Acids Res 41:W492-8