Theo Nikiforov

Fluorogenic polymerase, endonuclease, and ligase assays based on DNA substrates labeled with a single fluorophore

This paper describes the development of homogeneous, fluorogenic polymerase, restriction endonuclease, and ligase assays based on the use of DNA substrate molecules labeled with a single fluorophore. All three enzymatic assays are based on the same observed phenomenon whereby the fluorescence intensity of hairpin-type oligonucleotides with a 5single-stranded extension, labeled with a single fluorophore, changes when the distance of the dye from the 3 end of the molecule is altered as a result of the enzymatic transformation (i.e., polymerase extension, endonuclease hydrolysis, or ligation). The magnitudes of the observed fluorescence intensity changes range from 1.2-fold to 3.9-fold, and they are dependent on the type of dye used, its position within the substrate and product molecules, and the base composition surrounding the labeling site.

Fluorogenic substrates with single fluorophores for nucleic acid-modifying enzymes: Design principles and new applications

Nucleic acid-modifying enzymes are widely used in numerous applications. Many of these proteins are also important drug targets. Thus, better assays for the evaluation of their activities are always needed and are continuously being developed. Recently, I reported on a set of assays for several DNA-modifying enzymes (polymerases, endonucleases, and ligase) based on simple, hairpin-type oligonucleotide substrates labeled with a single fluorophore (Anal. Biochem. 412 (2011) 229-236). The present paper reports further studies on the mechanism of action of these substrates. It was assumed that the single fluorophore of these substrates is substantially quenched by stacking onto the terminal base(s) of the duplex, and that any perturbation of that stacking causes an increase in fluorescence. Based on this assumption, substrates of the same type for a variety of additional enzymes were developed and tested. The new assays described herein are for T4 polynucleotide kinase, the DNA repair enzymes uracil-DNA glycosylase (UDG) and formamido-pyrimidine-DNA glycosylase (FPG), 3-5 exonucleases, and enzymes with template-independent terminal transferase activity such as Taq polymerase. All of these molecules are easy to synthesize, and similar substrates for other enzymes can rapidly be designed based on the principles outlined in this work.

Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases

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.