Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems. 1. Introduction The design of tools for point mutations (SNP, one nucleotide insertion, or deletion) detection is a problem of current importance. Recently, various techniques for genetic diagnostics have been developed [1–3]. A large part of these methods is based on hybridization of fluorescent oligonucleotide probe to its complementary DNA target to generate fluorescent signal. Pyrene conjugates of oligonucleotides as potential diagnostic probes attract attention of researchers due to the unique properties of pyrene such as long fluorescence lifetime, considerable sensitivity to the microenvironment, high quantum yield, and ability to form excimers and exciplexes [4]. Earlier, tandems of monopyrene-coupled oligonucleotides have been designed for nucleic acids probing [5–7] and SNP diagnostics [8–11]. The principle of action of these probes is based on the excimer formation at the tandem junction where a pyrene unit on the 3′-terminus of one component of the probe interacts with a pyrene unit on the 5′-terminus of another component. Tandem excimer probes are of particular interest in case of detection of such subtle structural alterations caused by point mutations in DNA-tandem complexes. In contrast to tandem probes relying on fluorescence resonance energy transfer (FRET) [12] which is realized within 1–10？nm, excimer tandem probes rely on formation of excimer between two parallel pyrene units at a distance of only 3.4？？ which almost corresponds to the length of the one base pair along the DNA strand. We have been
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