Bleaching-resistant, near-continuous single-molecule fluorescence and FRET based on fluorogenic and transient DNA binding

Abstract

Photobleaching of fluorescent probes limits the observation span of a typical single molecule fluorescence measurement and hinders simultaneous observation of dynamics across timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes throughout the experiment via transient binding of fluorescently labelled single-stranded DNAs to complementary target DNA strands attached to the target molecule. We show our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the photobleaching time under our conditions. We also show our method is adaptable to FRET and study the conformational dynamics of DNA Holliday Junctions for extended periods. By adjusting the temporal resolution and observation span, we envision to capture the conformational dynamics of proteins and nucleic acids over wide range of timescales.

iMAXFRET (Information Maximized FRET) for multipoint single-molecule structural analysis

“…iMAX FRET has many advantages over established techniques. a) Short time required for measurement : As we use the stochastic exchange scheme for probing all possible points in a molecule with otherwise identical probes, this one-pot method cuts down the imaging time considerably as compared to other DNA hybridization-based imaging techniques 19,23,36 . The probe-labeled samples can be practically prepared in 24 hours 19 while weak-binder based fluorescence measurement takes as little as two minutes.…”

“…However, due to the complexity of signals, only one or two dye-labeled points in a single molecule can be tracked at a time 16 , precluding a comprehensive understanding of the three-dimensional perspective on the structure without prior knowledge of the molecular structure obtained by other means. Extensions of conventional smFRET, which allowed observation of multiple distances between a single reference point and several other positions in a molecule of interest using a scheme of DNA exchange, were developed by several groups [17][18][19] . Although this multi-point analysis mitigated the limitation of the conventional smFRET, the necessity of a single reference point still implied that positions in three-dimensional space could not be triangulated for de novo structural reconstruction.…”

“…Extensions of conventional smFRET, which allowed observation of multiple distances between a single reference point and several other positions in a molecule of interest using a scheme of DNA exchange, were developed by several groups 17-19 . Although this multi-point analysis mitigated the limitation of the conventional smFRET, the necessity of a single reference point still implied that positions in three-dimensional space could not be triangulated for de novo structural reconstruction.…”

Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions

“…As multiple imager strands can thus bind simultaneously and are designed to exchange on a time scale similar to that of dye photobleaching, this allowed us to follow the motion of DNA-origami on a supported lipid bilayer (SLB) for minutes rather than seconds 8 . While the concept of a continuous turnover of fluorophores to circumvent photobleaching has gained traction in the field of single-molecule fluorescence 11,12 , it is yet to be implemented in more complex biological samples.Here, we introduce a motif for dual-color DNA-PAINT-SPT for measuring protein-protein interactions at the single-molecule level, and use it to reliably quantify ligand-induced protein dimerization in membranes. We further extend our dual-color DNA-PAINT-SPT implementation to live cell imaging applications and demonstrate its improved performance over single-dye SPT.…”

DNA-PAINT single-particle tracking (DNA-PAINT-SPT) enables extended single-molecule studies of membrane protein interactions

“…As multiple imager strands can thus bind simultaneously and are designed to exchange on a time scale similar to that of dye photobleaching, this allowed us to follow the motion of DNA-origami on a supported lipid bilayer (SLB) for minutes rather than seconds 8 . While the concept of constantly exchanging fluorophores to prevent photobleaching has gained traction in the field of single-molecule fluorescence 11,12 , it is yet to be implemented in more complex biological samples.…”