Combined optical trapping and sm-FRET for nucleic acid folding studies

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By studying ancient RNA motifs found in all kingdoms of life, we are also looking back in time to a common evolutionary ancestor. Riboswitches and ribozymes are structured RNAs capable of gene regulation and catalysis. Their surprising catalytic/functional diversity and prevalence across species hints at an "RNA World" that predates our current paradigm which depends on DNA for information and protein for catalysis. The TPP riboswitch is an ancient ligand-binding RNA that regulates vitamin B1 (TPP) synthesis in bacteria, archaea, and even higher organisms like plants. Using single molecule optical trapping (OT) and the judicious application of piconewton forces, we have studied the folding pathways of the riboswitch and its response to TPP and other analogues. Ligand binding appears to form in two steps: TPP must first bind weakly before proceeding to a strongly bound state, as measured by the non-equilibrium work required to unfold it. To probe the physical origin of these states, we use a novel dual-beam optical trap combined with simultaneous FRET that can measure two orthogonal signals of the molecule's conformation. We demonstrate the direct observation of both the unfolding of secondary (base-paired) structures (via OT) and long-range tertiary contacts (via FRET) that form during ligand binding. In time, we anticipate this technique will become a sought-after tool that can expose the intricate folding pathways of complex biomolecules.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2014
Issuance monographic
Language English


Associated with Perez, Christian Francisco
Associated with Stanford University, Department of Physics.
Primary advisor Block, Steven M
Thesis advisor Block, Steven M
Thesis advisor Das, Rhiju
Thesis advisor Doniach, S
Advisor Das, Rhiju
Advisor Doniach, S


Genre Theses

Bibliographic information

Statement of responsibility Christian Francisco Perez.
Note Submitted to the Department of Physics.
Thesis Thesis (Ph.D.)--Stanford University, 2014.
Location electronic resource

Access conditions

© 2014 by Christian Francisco Perez
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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