High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

Abstract

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object. The limited number of available FRET pair fluorophores and complicated data analysis makes it challenging to apply single-molecule FRET for structural analysis of biomolecules. Currently, only a couple of FRET pairs can be reliably measured on a single object. Here we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the FRET efficiency and pair distance with sub-nanometer resolution. We determine the distance between other pairs by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. We envision that our FRET X technology will be a tool for the high-resolution structural analysis of biomolecules and other nano-structures.

The emerging landscape of single-molecule protein sequencing technologies

“…A different approach that allows for global pairwise distance measurements combines DNA technology with single-molecule Förster resonance energy transfer (FRET) 50 . The current state of the art for single-molecule FRET analysis allows only one or two FRET pairs to be probed at a time 51 , and new high-resolution FRET using transient binding between DNA tags allows for one FRET pair to be probed at a time while many probes are collectively present on a single protein 50 . Similarly to the approaches described above, specific amino acids (for example, lysine, cysteine, etc.)…”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…The fluorescence lifetimes and the correlations of fluorophores can be improved with a high temporal resolution, and comprehensive exhibition of the bioprocess can be obtained through the complex labeling strategies. By detecting several pairs of fluorophores in a nucleic acid complex , Filius et al [ 59 ] proposed “FRET X”, in which the detection of smFRET was sequentially done by hybridizing and exciting one donor-contained primer at a time ( Figure 4 B), and the dwell time was prolonged. The multiple labeling points and sequential signal detecting improved the accuracy of structure determination and can be coupled with the multiple virtual barcodes, developed by the coworker Kim et al [ 60 ] to observe multiple orthogonal probes in a short time.…”

Evaluation of FRET X for Single-Molecule Protein Fingerprinting

“…We have shown that FRET X can resolve the distance between multiple FRET pairs with sub-nanometer accuracy. 15 Here, we apply FRET X for protein fingerprinting. By detecting target amino acids one by one, FRET X produces a unique fingerprint, allowing identification of the protein from a reference database.…”