A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3′-OH unprotected cleavable fluorescent 2′-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor® 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA template

A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis†

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3′-OH unprotected cleavable fluorescent 2′-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor® 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal–no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates

Mapping the Antagonist Binding Site of the Serotonin Type 3 Receptor by Fluorescence Resonance Energy Transfer

We have measured fluorescence resonance energy transfer (FRET) between a fluorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the micelle surrounding the detergent-solubilized receptor. The exptl. obsd. FRET efficiency was evaluated on the basis of the characteristic dimensions of the receptor-micelle complex and the av. no. of acceptor mols. in such micelles. The binding site was detd. to be 5.4 +- 0.9 nm above the center of the detergent micelle. The expts. were performed below the crit. micellar concn. of the detergent (C12E9) used to solubilize the receptor, under which conditions it was demonstrated that the ligand binding activity was fully preserved. This reduces considerably the fluorescence background arising from probes not assocd. with the receptor, allowing a precise detn. of the transfer efficiency. [on SciFinder (R)

Fluorescence techniques for fundamental and applied studies of membrane protein receptors: the 5-HT3 serotonin receptor

A fluorescently labeled ligand for the 5-HT3 serotonin receptor was synthesized and its sub-nanomolar affinity for the purified, detergent solubilized receptor was measured. The change in the ligand's fluorescence upon receptor binding was used to directly measure its dissocn. const. for receptor binding, to det. the pharmacol. of the receptor, and finally to characterize the binding site of the receptor. A total internal reflection fluorescence (TIRF) assay for the 5-HT3 receptor was developed, which is suitable for high-through-put screening. Therefore, the receptor was immobilized via its C-terminal His-tag onto a nitrilotriacetic acid-modified quartz surface. The affinities of both the fluorescent ligand and several non-fluorescent compds. were rapidly detd. by the TIRF assay, and were shown to agree well with both the soln. and classical radioligand binding assays. This indicated that the functional integrity of the receptor was preserved at the sensor surface. Due to the extreme sensitivity, the TIRF assay allows a complete pharmacol. affinity profile of a quantity of receptor provided by a small no. of highly-expressing cells. [on SciFinder (R)