PT  - JOURNAL ARTICLE
AU  - Mirjam Kümmerlin
AU  - Abhishek Mazumder
AU  - Achillefs N. Kapanidis
TI  - Bleaching-resistant, near-continuous single-molecule fluorescence and FRET based on fluorogenic and transient DNA binding
AID  - 10.1101/2022.04.19.488730
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.04.19.488730
4099  - http://biorxiv.org/content/early/2022/12/06/2022.04.19.488730.short
4100  - http://biorxiv.org/content/early/2022/12/06/2022.04.19.488730.full
AB  - 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 should enable capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.Competing Interest StatementThe work was performed using miniaturized commercial microscopes from Oxford Nanoimaging, a company in which Achillefs N Kapanidis is a co-founder and shareholder.