Kummerlin, M. | bioRxiv Science

bioRxiv2023

Engineering Modular and Tunable Single Molecule Sensors by Decoupling Sensing from Signal Output

Biosensors play key roles in medical research and diagnostics, but there currently is a lack of sensing platforms that combine easy adaptation to new targets, strategies to tune the response window to relevant analyte concentration ranges and allow for the incorporation of multiple sensing elements to benefit from multivalency. Utilizing a DNA origami nanostructure as a scaffold for arranging the different sensor components, we here propose an approach for the development of modular and tunable single-molecule sensors capable of detecting a variety of biomolecular targets such as nucleic acids, antibodies and restriction enzymes while offering mechanisms to tune the dynamic window, the specificity, and the cooperativity of the sensor.

biophysics↗

bioRxiv2022

Bleaching-resistant single-molecule fluorescence and FRET monitoring based on fluorophore exchange via transient DNA binding

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 Forster 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.

biophysics↗