Direct observation of the interaction of single fluorescent nucleotide analogue molecules with DNA polymerase I

The interaction of a fluorescent nucleotide analogue, 2′- (or-3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP), with the Klenow fragment of DNA polymerase I (Pol I) was visualized at a single-molecule level. Upon excitation, individual enzyme–TNP–ATP complexes resulted in bright fluorescent spots owing to the increase of the fluorescence quantum yield of TNP–ATP when it bound to the enzyme molecule, whereas unbound TNP–ATP molecules were not visible in the single molecule detection. Thus, we directly investigated the individual interactions of TNP–ATP with the enzyme using single-molecule fluorescence imaging and time-resolved spectroscopy of single enzyme–TNP–ATP complexes without prior separation of the unbound probe molecules.