Recent advances in fluorogen-binding “light-up” RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2,
Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins
- NSF-PAR ID:
- 10452988
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The FEBS Journal
- Volume:
- 288
- Issue:
- 6
- ISSN:
- 1742-464X
- Page Range / eLocation ID:
- p. 1742-1758
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract i MangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding ini MangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications. -
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