Multimodal measurements of single-molecule conformational dynamics
Abstract/Contents
- Abstract
- Single-molecule methods can monitor the dynamics of biological macromolecules in real time. Here we develop FluoRBT, which provides multimodal measurements of structural transitions in nucleic acids and nucleoprotein complexes by combining magnetic tweezers manipulation, infrared darkfield imaging, and single-molecule FRET. We demonstrate the capabilities of FluoRBT by observing structural transitions in DNA under mechanical strain through concurrent changes in FRET and angle or extension. We also apply FluoRBT to study the dynamics of DNA interrogation by the RNA-guided CRISPR nuclease Cas9. We identify a structural intermediate corresponding to DNA:RNA hybridization within the target "seed" region, and characterize how the interplay between sequence mismatches and DNA superhelicity modulates access to states competent for stable binding and cleavage. FluoRBT can further be used to dissect the mechanism of mechanochemical coupling in DNA gyrase by correlating progress through the supercoiling reaction with rich dynamics of the N-gate ATPase domain observed in single-molecule FRET assays. By combining mechanical measurements of global deformations in nucleic acids with FRET measurements of local conformational changes, FluoRBT enables multidimensional investigations of systems ranging from DNA structures to large molecular machines.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Ivanov, Ivan Enchev |
|---|---|
| Degree supervisor | Bryant, Zev David |
| Degree supervisor | Spakowitz, Andrew James |
| Thesis advisor | Bryant, Zev David |
| Thesis advisor | Spakowitz, Andrew James |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | De la Zerda, Adam |
| Degree committee member | Khosla, Chaitan, 1964- |
| Degree committee member | De la Zerda, Adam |
| Associated with | Stanford University, Department of Chemical Engineering. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Ivan Enchev Ivanov. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by Ivan Enchev Ivanov
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