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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM102519-01
Application #
8345532
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2012-09-30
Project End
2017-07-31
Budget Start
2012-09-30
Budget End
2013-07-31
Support Year
1
Fiscal Year
2012
Total Cost
$298,300
Indirect Cost
$108,300
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Tian, Siqi; Das, Rhiju (2016) RNA structure through multidimensional chemical mapping. Q Rev Biophys 49:e7
Chou, Fang-Chieh; Kladwang, Wipapat; Kappel, Kalli et al. (2016) Blind tests of RNA nearest-neighbor energy prediction. Proc Natl Acad Sci U S A 113:8430-5
Tian, Siqi; Yesselman, Joseph D; Cordero, Pablo et al. (2015) Primerize: automated primer assembly for transcribing non-coding RNA domains. Nucleic Acids Res 43:W522-6
Miao, Zhichao; Adamiak, Ryszard W; Blanchet, Marc-Frédérick et al. (2015) RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures. RNA 21:1066-84
Cheng, Clarence Yu; Chou, Fang-Chieh; Das, Rhiju (2015) Modeling complex RNA tertiary folds with Rosetta. Methods Enzymol 553:35-64
Cordero, Pablo; Das, Rhiju (2015) Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis. PLoS Comput Biol 11:e1004473
Xue, Shifeng; Tian, Siqi; Fujii, Kotaro et al. (2015) RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation. Nature 517:33-8
Cheng, Clarence Yu; Chou, Fang-Chieh; Kladwang, Wipapat et al. (2015) Consistent global structures of complex RNA states through multidimensional chemical mapping. Elife 4:e07600
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

Showing the most recent 10 out of 14 publications