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, which makes
it difficult to apply single-molecule FRET for structural analysis
of biomolecules. 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
pair distance with subnanometer precision. The distance between other
pairs are determined by sequentially exchanging DNA strands. We name
this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET
X technology will be a tool for the high-resolution analysis of biomolecules
and nanostructures.
Detection of specific nucleic acid sequences is invaluable in biological studies such as genetic disease diagnostics and genome profiling. Here, we developed a highly sensitive and specific detection method that combines an advanced oligonucleotide ligation assay with multicolor single-molecule fluorescence. We demonstrated that under our experimental conditions, 7-nucleotide long DNA barcodes have the optimal short length to ascertain specificity while being long enough for sufficient ligation. Using four spectrally separated fluorophores to label DNA barcodes, we simultaneously distinguished four DNA target sequences differing by only a single nucleotide. Our single-molecule approach will allow for accurate identification of low-abundance molecules without the need for target DNA preamplification.
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.
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