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.

Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores.

We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer.

Application of fluorescence polarization to enzyme assays and single nucleotide polymorphism genotyping: some recent developments.

This article describes some recent developments in the field of fluorescence polarization (FP) as applied to enzyme assays and single nucleotide polymorphism (SNP) genotyping. First, we present our recent progress on the application of fluorescence polarization to high throughput screening (HTS). We show how the use of a 2-thiopyridinone-based, mixed disulfide biotinylation reagent can shorten the assay time of our recently reported kinase assay method based on thiophosphorylation and biotinylation from several hours to a few minutes.

Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection.

Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping.

Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase, and protease reactions.

We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides kinases, such as phosphatases and proteases. By adjustment of the ionic strength of the buffer it is possible to use this method in cases where both the substrate and the enzymatic product are highly negatively charged.