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 fragme nt of DNA polymerase I (Pol I) was visualized at a single-molecule level. U pon excitation, individual enzyme-TNP-ATP complexes resulted in bright fluo rescent spots owing to the increase of the fluorescence quantum yield of TN P-ATP when it bound to the enzyme molecule, whereas unbound TNP-ATP molecul es were not visible in the single molecule detection, Thus, we directly inv estigated the individual interactions of TNP-ATP with the enzyme using sing le-molecule fluorescence imaging and time-resolved spectroscopy of single e nzyme-TNP-ATP complexes without prior separation of the unbound probe molec ules. (C) 2000 Elsevier Science B.V. All rights reserved.