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,
G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.
more » « less- NSF-PAR ID:
- 10090060
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- Article No. 201815162
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract A human telomere ends in a single-stranded 3′ tail, composed of repeats of T2AG3. G-quadruplexes (GQs) formed from four consecutive repeats have been shown to possess high-structural and mechanical diversity. In principle, a GQ can form from any four repeats that are not necessarily consecutive. To understand the dynamics of GQs with positional multiplicity, we studied five and six repeats human telomeric sequence using a combination of single molecule FRET and optical tweezers. Our results suggest preferential formation of GQs at the 3′ end both in K+ and Na+ solutions, with minor populations of 5′-GQ or long-loop GQs. A vectorial folding assay which mimics the directional nature of telomere extension showed that the 3′ preference holds even when folding is allowed to begin from the 5′ side. In 100 mM K+, the unassociated T2AG3 segment has a streamlining effect in that one or two mechanically distinct species was observed at a single position instead of six or more observed without an unassociated repeat. We did not observe such streamlining effect in 100 mM Na+. Location of GQ and reduction in conformational diversity in the presence of extra repeats have implications in telomerase inhibition, T-loop formation and telomere end protection.more » « less
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Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔ
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