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.
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