Single-molecule fret for protein characterization

Summary

The invention provides an analysis method (100) for characterization of a tagged protein (10) using FRET donor-acceptor pair chromophores (20), wherein the FRET donor- acceptor pair chromophores (20) comprise a first chromophore (21) and a second chromophore (22), wherein the FRET donor-acceptor pair chromophores (20) have a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range, wherein one of the FRET donor-acceptor pair chromophores (23) is excitable by donor excitation radiation (51) in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair chromophores (24) is configured to provide acceptor emission radiation (54) in the acceptor emission radiation range upon excitation with donor excitation radiation (51) in the donor excitation radiation range of the one of the FRET donor-acceptor pair chromophores (23) when the first chromophore (21) and the second chromophore (22) are configured within a predetermined distance, wherein the tagged protein (10) comprises a first amino acid (11) tagged with a first tag (31) and a second amino acid (12) tagged with a second tag (32), wherein the first tag (31) comprises the first chromophore (21) or is associated to the first chromophore (21), wherein the second tag (32) comprises an oligonucleotide, and wherein the analysis method (100) comprises: a barcode exposure stage (110) comprising: (i) exposing the tagged protein (10) to a barcode (42), wherein the barcode (42) is configured to hybridize with the second tag (32), and wherein the barcode (42) comprises the second chromophore (22), (ii) providing radiation having a wavelength selected from the donor excitation radiation range to the tagged protein (10), and (iii) measuring emission in the donor emission radiation range and the acceptor emission radiation range to provide an emission signal.