Two successive reactions on a DNA template: a strategy for improving background fluorescence and specificity in nucleic acid detection.

We report a new strategy for template-mediated fluorogenic chemistry that results in enhanced performance for the fluorescence detection of nucleic acids. In this approach, two successive templated reactions are required to induce a fluorescence signal, rather than only one. These novel fluorescein-labeled oligonucleotide probes, termed 2-STAR (STAR = Staudinger-triggered α-azidoether release) probes, contain two quencher groups tethered by separate reductively cleavable linkers. When a 2-STAR quenched probe successively binds adjacent to two mono-triphenylphosphine-(TPP)-DNAs or one dual-TPP-DNA, the two quenchers are released, resulting in a fluorescence signal. Because of the requirement for two consecutive reactions, 2-STAR probes display an unprecedented level of sequence specificity for template-mediated probe designs. At the same time, background emission generated by off-template reactions or incomplete quenching is among the lowest of any fluorogenic reactive probes for the detection of DNA or RNA.