Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes.

Generic assay format for endo- and exonucleases based on fluorogenic substrates labeled with single fluorophores.

We previously described the development of fluorogenic assays for nucleic acid-modifying enzymes based on synthetic oligonucleotides labeled with a single fluorophore. In the current work, we studied the performance of such singly labeled substrates as a function of the nucleotide sequence in the vicinity of the fluorophore and the nature of the fluorophore itself. In agreement with published studies, we found that a 3' end of the primer terminating in a dC residue opposite a 5' dG provides the greatest degree of fluorophore quenching.

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.

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.

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 5'single-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.