Single-molecule, real-time measurement of enzyme kinetics by alternating-laser excitation fluorescence resonance energy transfer.

Using a single-molecule fluorescence method uniquely suitable for binding assay, alternating-laser excitation fluorescence resonance energy transfer (ALEX-FRET), we accurately measured the cleavage rate of 8-17 deoxyribozyme, an RNA-cleaving enzyme, at the single-molecule level in real time with a minimum consumption of samples, i.e., at least three orders of magnitude smaller than used in the conventional ensemble FRET method.