Journal article
Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding
- Abstract:
-
Graphical Abstract
A general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule is presented. Using two orthogonal sequences, the authors show that their method is adaptable to Förster resonance energy transfer (FRET) and can be used to continuously study the conformational transitions of dynamic structures for extended periods (>1 hr).
Abstract
Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
Actions
Access Document
- Files:
-
-
(Preview, Version of record, pdf, 1.9MB, Terms of use)
-
- Publisher copy:
- 10.1002/cphc.202300175
Authors
- Publisher:
- Wiley
- Journal:
- ChemPhysChem More from this journal
- Volume:
- 24
- Issue:
- 12
- Article number:
- e202300175
- Publication date:
- 2023-04-12
- Acceptance date:
- 2023-03-16
- DOI:
- EISSN:
-
1439-7641
- ISSN:
-
1439-4235
- Pmid:
-
37043705
- Language:
-
English
- Keywords:
- Pubs id:
-
1337552
- Local pid:
-
pubs:1337552
- Deposit date:
-
2023-06-02
Terms of use
- Copyright holder:
- Kümmerlin et al.
- Copyright date:
- 2023
- Rights statement:
- © 2023 The Authors. ChemPhysChem published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- Licence:
- CC Attribution (CC BY)
If you are the owner of this record, you can report an update to it here: Report update to this record