Encoding Multiple Virtual Signals in DNA Barcodes with Single-Molecule FRET

DNA barcoding provides a way to label myriad of different biological molecules using the extreme programmability in sequence synthesis. Fluorescence imaging is presumably most easy-to-access method for barcoding, yet large spectral overlaps between fluorescence dyes severely limit numbers barcodes that can be detected simultaneously. We here demonstrate use single-molecule resonance energy transfer (FRET) encode virtual signals conventional two-color microscopy. By optimizing and biochemistry conditions weak hybridization events, we markedly enhanced accuracy our determination FRET efficiency exhibited by each binding event barcode sequences. This allowed us unambiguously differentiate six encoding values without involving any probe exchanges. Our directly incorporated with previous techniques, may thus widely adopted expand signal space barcoding.