Target-assembled ExciProbes: application to DNA detection at the level of PCR product and plasmid DNA.

Recently, we introduced a novel exciplex-based approach for detection of nucleic acids using a model DNA-mounted exciplex system, consisting of two 8-mer ExciProbes hybridized to a complementary 16-mer DNA target. We now show, for the first time, that this approach can be used to detect DNA at the level of PCR product and plasmid, when the target sequence (5'-GCCAAACACAGAATCG-3') was embedded in long DNA molecules (PCR products and approximately 3 Kbp plasmid). A remarkably stringent demand is made of the solvent conditions for this exciplex emission to occur, viz., emission is optimal for DNA at 80% trifluoroethanol, even in the plasmid situations, raising the question of the molecular structural basis of this system. We show that a perfectly matched plasmid target can be differentiated from target containing single nucleotide substitutions; hence, ExciProbes could be applied to SNP analysis. The effect of counter cations (Na(+), K(+), and Mg(2+)) and PCR additives on exciplex emission has been also examined.

Detection of nucleic acids in situ: novel oligonucleotide analogues for target-assembled DNA-mounted exciplexes.

This research describes the effects of structural variation and medium effects for the novel split-oligonucleotide (tandem) probe systems for exciplex-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the exciplex partners and the consequent exciplex fluorescence emission on excitation. The model system consists of two 8-mer oligonucleotides, complementary to adjacent sites of a 16-mer DNA target. Each probe oligonucleotide is equipped with functions able to form an exciplex on correct, contiguous hybridization. This study investigates the influence of a number of structural aspects (i.e. chemical structure and composition of exciplex partners, length and structure of linker groups, locations of exciplex partner attachment, as well as effects of media) on the performance of DNA-mounted exciplex systems. The extremely rigorous structural demands for exciplex formation and emission required careful structural design of linkers and partners for exciplex formation, which are here described. Certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplexes, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. The exciplexes formed emitted at approximately 480 nm with large Stokes shifts ( approximately 130-140 nm). Comparative studies with pyrene excimer systems were also carried out.

Target-assembled tandem oligonucleotide systems based on exciplexes for detecting DNA mismatches and single nucleotide polymorphisms.

We report the first exciplex-based split-probe system for DNA detection. The detector is split at a molecular level into signal-silent components which, before a signal is generated, must be assembled correctly into a particular three-dimensional arrangement. The model system comprises of two 8-mer oligonucleotides, complementary to neighbouring sites of a 16-mer DNA target, each equipped with moieties able to form an exciplex on correct, contiguous hybridization. The exciplex emits at approximately 480 nm with a large Stokes shift (135 nm). The extremely rigorous structural demands for exciplex formation and emission were achieved by careful structural design and by the discovery that high levels of certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplex, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. Inserts and mismatches can be effectively detected by this exciplex construct giving potential for single nucleotide polymorphism detection.

An exciplex-based, target-assembled fluorescence system with inherently low background to probe for specific nucleic acid sequences.

A novel detection technique, called ExciProbes, has been developed to proof-of-principle level for DNA oligonucleotides. The new approach is based on the use of two short oligonucleotides complementary to a target nucleic acid sequence. Each short-probe oligonucleotide bears the separated parts of a new class of fluorescence detector, an exciplex. These isolated parts of the detector have no inherent signal at the detection wavelength. They are designed to detect biotarget by being assembled by the target itself to give a new molecular entity (the exciplex), with a characteristic fluorescence and very large Stokes shift (typically >150 nm). The technique is not related to fluorescence resonance energy transfer, and can potentially resolve to 1 base pair. ExciProbes can detect single or double mutations in a short sequence of DNA, and can be combined with temperature-filtering to provide allelic discrimination of single nucleotide polymorphism analysis. Compared to other fluorophore systems that have large backgrounds (typically >60%), ExciProbes show backgrounds of <1% under comparable conditions, and can be used with DNA, RNA, or synthetic nucleic acids such as locked nucleic acid.