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Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2',3'-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The fluorescence detection system for the analytical ultracentrifuge (AU-FDS) enables the measurement of hydrodynamic properties and interactions of biomolecules at subnanomolar concentrations. In this study, we describe methods for (i) preparing and purifying fluorescently labeled biomolecules and (ii) determining the meniscus position in the AU-FDS using BODIPY 493/503 fluorescent dye suspended in light oil. We subsequently use these methods to measure the interaction of DNA with Escherichia coli Klenow fragment (KF) and show that KF binds matched DNA to form 1:1 and 2:1 (protein/DNA) complexes with dissociation constants of 4.2 and 22 nM, respectively.