BTI1, an azoreductase with pH-dependent substrate specificity.

The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.

Time gating improves sensitivity in energy transfer assays with terbium chelate/dark quencher oligonucleotide probes.

Lanthanides are attractive as biolabels because their long luminescence decay rates allow time-gated detection, which separates background scattering and fluorescence from the lanthanide emission. A stable and highly luminescent terbium complex based on a tetraisophthalamide (TIAM) chelate is paired with a polyaromatic-azo dark quencher (referred to as a Black Hole Quencher or BHQ) to prepare a series of 5'TIAM(Tb)/3'BHQ dual-labeled oligonucleotide probes with no secondary structure. Luminescence quenching efficiency within terbium/BHQ probes is very dependent on the terbium-BHQ distance.

Intramolecular dimers: a new design strategy for fluorescence-quenched probes.

Fluorogenic probes dual-labeled with reporter and quencher dyes use a change in fluorescence to monitor biochemical events (e.g., substrate binding or enzyme digestion).

Evaluation of new linkers and synthetic methods for internal modified oligonucleotides.

Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine.

New reagents and methods for the synthesis of internal and 3'-labeled DNA.

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes.

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through Förster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.