Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding
<p><strong>Graphical Abstract</strong><br> <strong>A general strategy</strong> 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...
| Main Authors: | , , |
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| Format: | Journal article |
| Language: | English |
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Wiley
2023
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| _version_ | 1797110573345800192 |
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| author | Kümmerlin, M Mazumder, A Kapanidis, AN |
| author_facet | Kümmerlin, M Mazumder, A Kapanidis, AN |
| author_sort | Kümmerlin, M |
| collection | OXFORD |
| description | <p><strong>Graphical Abstract</strong><br>
<strong>A general strategy</strong> 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).<br><br>
<strong>Abstract</strong><br>
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.<br><br></p> |
| first_indexed | 2024-03-07T07:56:42Z |
| format | Journal article |
| id | oxford-uuid:e1b4baca-dbaa-4b94-92ec-addc0fa12f92 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:56:42Z |
| publishDate | 2023 |
| publisher | Wiley |
| record_format | dspace |
| spelling | oxford-uuid:e1b4baca-dbaa-4b94-92ec-addc0fa12f922023-08-21T09:52:28ZBleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA bindingJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:e1b4baca-dbaa-4b94-92ec-addc0fa12f92EnglishSymplectic ElementsWiley2023Kümmerlin, MMazumder, AKapanidis, AN<p><strong>Graphical Abstract</strong><br> <strong>A general strategy</strong> 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).<br><br> <strong>Abstract</strong><br> 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.<br><br></p> |
| spellingShingle | Kümmerlin, M Mazumder, A Kapanidis, AN Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding |
| title | Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding |
| title_full | Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding |
| title_fullStr | Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding |
| title_full_unstemmed | Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding |
| title_short | Bleaching-resistant,near-continuous single-molecule fluorescence and fret based on fluorogenic and transient DNA binding |
| title_sort | bleaching resistant near continuous single molecule fluorescence and fret based on fluorogenic and transient dna binding |
| work_keys_str_mv | AT kummerlinm bleachingresistantnearcontinuoussinglemoleculefluorescenceandfretbasedonfluorogenicandtransientdnabinding AT mazumdera bleachingresistantnearcontinuoussinglemoleculefluorescenceandfretbasedonfluorogenicandtransientdnabinding AT kapanidisan bleachingresistantnearcontinuoussinglemoleculefluorescenceandfretbasedonfluorogenicandtransientdnabinding |