Advancing multidimensional chemical mapping of RNA structure through next-generation sequencing
- RNAs are an essential and versatile category of biopolymers, fulfilling roles from relaying genetic information to catalysis to regulation of gene expression. The formation of and interconversion between conformational states is crucial to the biological functions of RNAs. Chemical mapping methods have proven valuable in providing solution-state, per-nucleotide structural information for RNAs, and computational prediction methods, which can incorporate chemical mapping data, have permitted modeling of both secondary and tertiary RNA structures. However, certain techniques have faced limitations: high-confidence secondary structure prediction through '2D' chemical mapping of systematic mutants (mutate-and-map, M2) is limited by RNA size, and tertiary structure inference through analysis of spatially localized hydroxyl radicals (multiplexed •OH cleavage analysis with gel electrophoresis, MOHCA-gel) is limited by imprecise readouts of proximal residue pairs. Next-generation sequencing (NGS) has greatly enhanced the throughput of chemical mapping, allowing for estimation of chemical reactivities for viral genomes and cellular transcriptomes, but has not yet been leveraged to alleviate the limitations on M2 and MOHCA. In this dissertation, I present the application of NGS readouts to advance a multidimensional chemical mapping (MCM) pipeline for probing and modeling RNA secondary and tertiary structure. First, I demonstrate that a sequencing-based MOHCA-seq method allows for precise detection of tertiary proximities throughout target non-coding RNA (ncRNA) domains. Using constraints from MOHCA-seq data improves Rosetta modeling with experimentally inferred secondary structures to 1-nm accuracy, which allows both visualization of complex ncRNA states, such as the ligand-free states of cyclic-di-GMP, glycine, and adenosylcobalamin riboswitch aptamers, and blind structure predictions of a 188-nt lariat capping ribozyme and a Hox internal ribosome entry site domain. Second, I apply MCM to a bioengineered, directionally switchable RNA-protein motor and demonstrate that it is able to detect distinct sets of tertiary proximities in alternative conformations of a multistate RNA system. Finally, I show that the length and throughput limits of M2 can be overcome by deriving 2D data from high-modification mutational profiling experiments, which permit readout of multiple modifications in single RNA molecules, and explore the basis of the 2D signal. These results improve the applicability and usability of the MCM pipeline and enable structural characterization of previously intractable RNAs.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Cheng, Clarence Y
|Stanford University, Department of Biochemistry.
|Straight, Aaron, 1966-
|Straight, Aaron, 1966-
|Statement of responsibility
|Clarence Y. Cheng.
|Submitted to the Department of Biochemistry.
|Thesis (Ph.D.)--Stanford University, 2016.
- © 2016 by Clarence Yu Cheng
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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