Modeling and validation of RNA secondary structures and ensembles through multidimensional chemical mapping
Abstract/Contents
- Abstract
- Ribonucleic acid (RNA) molecules play important roles in many of the core processes of life. The versatility of RNA functions relies on its ability to adopt and interconvert between distinct folding states. However, RNA conformations have largely eluded experimental dissection and validation due to the lack of generally applicable quantitative methods. In this dissertation, I present the recent development of the lock-mutate-map-rescue (LM2R) workflow, which leverages systematic mutagenesis and high-throughput RNA chemical mapping to unambiguously validate or falsify base-pairing models. First, the mutate-map-rescue (M2R) method was established on a small bacterial 16S ribosomal RNA domain, provided incisive evidence falsifying a prior SHAPE-derived model, and instead revealed a structural equilibrium between a crystallographic secondary structure and one with a single-nucleotide register shift. After the initial success, I applied M2R to two novel real-world RNA regulatory elements that possess crucial biological functions. The relevance of M2R-validated secondary structure models was further strengthened by in vivo assays, fueling discoveries of new paradigms in biology. Furthermore, to dissect the solution structures of a paradoxical adenine riboswitch, I advanced the technique into LM2R with an additional layer of mutagenesis to obtain 'locking' mutants that each isolates a single state. With such stabilizers enabling rigorous tests for co-existence of helices within a particular state, LM2R gave rise to a unified ensemble model that resolves discrepancies between previous alternative models. Finally, I made available an algorithm that optimizes PCR assembly design for RNA preparation, and created the Primerize-2D server that greatly simplifies LM2R library synthesis. In sum, as the central component of the multidimensional chemical mapping (MCM) pipeline, LM2R provides a general and practical route to robust validation of RNA secondary structures and ensembles.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Tian, Siqi | |
|---|---|---|
| Associated with | Stanford University, Department of Biochemistry. | |
| Primary advisor | Das, Rhiju | |
| Thesis advisor | Das, Rhiju | |
| Thesis advisor | Greenleaf, William James | |
| Thesis advisor | Herschlag, Daniel | |
| Advisor | Greenleaf, William James | |
| Advisor | Herschlag, Daniel |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Siqi Tian. |
|---|---|
| Note | Submitted to the Department of Biochemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Siqi Tian
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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