The potential of fluorogenicity for single molecule FRET and DyeCycling
Single Molecule Förster Resonance Energy Transfer (smFRET) is a popular technique to directly observe biomolecular dynamics in real time, offering unique mechanistic insight into proteins, ribozymes, and so forth. However, inevitable photobleaching of the fluorophores puts a stringent limit on the t...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Cambridge University Press
2024-01-01
|
| Series: | QRB Discovery |
| Subjects: | |
| Online Access: | https://www.cambridge.org/core/product/identifier/S2633289224000115/type/journal_article |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832542439962836992 |
|---|---|
| author | Srijayee Ghosh Sonja Schmid |
| author_facet | Srijayee Ghosh Sonja Schmid |
| author_sort | Srijayee Ghosh |
| collection | DOAJ |
| description | Single Molecule Förster Resonance Energy Transfer (smFRET) is a popular technique to directly observe biomolecular dynamics in real time, offering unique mechanistic insight into proteins, ribozymes, and so forth. However, inevitable photobleaching of the fluorophores puts a stringent limit on the total time a surface-tethered molecule can be monitored, fundamentally limiting the information gain through conventional smFRET measurements. DyeCycling addresses this problem by using reversibly – instead of covalently – coupled FRET fluorophores, through which it can break the photobleaching limit and theoretically provide unlimited observation time. In this perspective paper, we discuss the potential of various fluorogenic strategies to suppress the background fluorescence caused by unbound, freely diffusing fluorophores inherent to the DyeCycling approach. In comparison to nanophotonic background suppression using zero-mode waveguides, the fluorogenic approach would enable DyeCycling experiments on regular glass slides with fluorogenic FRET probes that are quenched in solution and only fluoresce upon target binding. We review a number of fluorogenic approaches and conclude, among other things, that short-range quenching appears promising for realising fluorogenic DyeCycling on regular glass slides. We anticipate that our discussion will be relevant for all single-molecule fluorescence techniques that use reversible fluorophore binding. |
| format | Article |
| id | doaj-art-49f430f325df48dd975f7e66046bcbfb |
| institution | Kabale University |
| issn | 2633-2892 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Cambridge University Press |
| record_format | Article |
| series | QRB Discovery |
| spelling | doaj-art-49f430f325df48dd975f7e66046bcbfb2025-02-04T04:10:13ZengCambridge University PressQRB Discovery2633-28922024-01-01510.1017/qrd.2024.11The potential of fluorogenicity for single molecule FRET and DyeCyclingSrijayee Ghosh0https://orcid.org/0000-0003-2869-1993Sonja Schmid1https://orcid.org/0000-0002-3710-5602Department of Chemistry, University of Basel, Basel, SwitzerlandDepartment of Chemistry, University of Basel, Basel, SwitzerlandSingle Molecule Förster Resonance Energy Transfer (smFRET) is a popular technique to directly observe biomolecular dynamics in real time, offering unique mechanistic insight into proteins, ribozymes, and so forth. However, inevitable photobleaching of the fluorophores puts a stringent limit on the total time a surface-tethered molecule can be monitored, fundamentally limiting the information gain through conventional smFRET measurements. DyeCycling addresses this problem by using reversibly – instead of covalently – coupled FRET fluorophores, through which it can break the photobleaching limit and theoretically provide unlimited observation time. In this perspective paper, we discuss the potential of various fluorogenic strategies to suppress the background fluorescence caused by unbound, freely diffusing fluorophores inherent to the DyeCycling approach. In comparison to nanophotonic background suppression using zero-mode waveguides, the fluorogenic approach would enable DyeCycling experiments on regular glass slides with fluorogenic FRET probes that are quenched in solution and only fluoresce upon target binding. We review a number of fluorogenic approaches and conclude, among other things, that short-range quenching appears promising for realising fluorogenic DyeCycling on regular glass slides. We anticipate that our discussion will be relevant for all single-molecule fluorescence techniques that use reversible fluorophore binding.https://www.cambridge.org/core/product/identifier/S2633289224000115/type/journal_articlefluorescenceFRETfluorogenicconformational dynamicssingle molecule kinetics |
| spellingShingle | Srijayee Ghosh Sonja Schmid The potential of fluorogenicity for single molecule FRET and DyeCycling QRB Discovery fluorescence FRET fluorogenic conformational dynamics single molecule kinetics |
| title | The potential of fluorogenicity for single molecule FRET and DyeCycling |
| title_full | The potential of fluorogenicity for single molecule FRET and DyeCycling |
| title_fullStr | The potential of fluorogenicity for single molecule FRET and DyeCycling |
| title_full_unstemmed | The potential of fluorogenicity for single molecule FRET and DyeCycling |
| title_short | The potential of fluorogenicity for single molecule FRET and DyeCycling |
| title_sort | potential of fluorogenicity for single molecule fret and dyecycling |
| topic | fluorescence FRET fluorogenic conformational dynamics single molecule kinetics |
| url | https://www.cambridge.org/core/product/identifier/S2633289224000115/type/journal_article |
| work_keys_str_mv | AT srijayeeghosh thepotentialoffluorogenicityforsinglemoleculefretanddyecycling AT sonjaschmid thepotentialoffluorogenicityforsinglemoleculefretanddyecycling AT srijayeeghosh potentialoffluorogenicityforsinglemoleculefretanddyecycling AT sonjaschmid potentialoffluorogenicityforsinglemoleculefretanddyecycling |