Backer, AS;
King, GA;
Biebricher, AS;
Shepherd, JW;
Noy, A;
Leake, MC;
Heller, I;
... Peterman, EJG; + view all
(2021)
Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy.
Journal of Physical Chemistry B
, 125
(30)
pp. 8351-8361.
10.1021/acs.jpcb.1c02708.
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Abstract
The combination of DNA force spectroscopy and polarization microscopy of fluorescent DNA intercalator dyes can provide valuable insights into the structure of DNA under tension. These techniques have previously been used to characterize S-DNA—an elongated DNA conformation that forms when DNA overstretches at forces ≥ 65 pN. In this way, it was deduced that the base pairs of S-DNA are highly inclined, relative to those in relaxed (B-form) DNA. However, it is unclear whether and how topological constraints on the DNA may influence the base-pair inclinations under tension. Here, we apply polarization microscopy to investigate the impact of DNA pulling geometry, torsional constraint, and negative supercoiling on the orientations of intercalated dyes during overstretching. In contrast to earlier predictions, the pulling geometry (namely, whether the DNA molecule is stretched via opposite strands or the same strand) is found to have little influence. However, torsional constraint leads to a substantial reduction in intercalator tilting in overstretched DNA, particularly in AT-rich sequences. Surprisingly, the extent of intercalator tilting is similarly reduced when the DNA molecule is negatively supercoiled up to a critical supercoiling density (corresponding to ∼70% reduction in the linking number). We attribute these observations to the presence of P-DNA (an overwound DNA conformation). Our results suggest that intercalated DNA preferentially flanks regions of P-DNA rather than those of S-DNA and also substantiate previous suggestions that P-DNA forms predominantly in AT-rich sequences.
| Type: | Article |
|---|---|
| Title: | Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1021/acs.jpcb.1c02708 |
| Publisher version: | https://doi.org/10.1021/acs.jpcb.1c02708 |
| Language: | English |
| Additional information: | © 2021 The Authors. Published by American Chemical Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| Keywords: | Science & Technology, Physical Sciences, Chemistry, Physical, Chemistry, SINGLE-STRANDED-DNA, OPTICAL TWEEZERS, MELTING BUBBLES, PEELED SSDNA, S-DNA, ORIENTATION, MOLECULES, DYNAMICS, INTERCALATION, COMPETITION |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10133906 |
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