Abstract: Triplex complexes contain a single-stranded probe bound to a double-stranded nucleic acid target, in which the probe includes a heteropolymeric nucleic acid or a heteropolymeric nucleic acid analog. All base triplets of the complex are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C. A cation-facilitated assay includes detecting the presence of such triplex complexes to determine the degree of complementarity between the probe and target sequence. The assay preferably detects a change in fluorescent intensity of a label as a function of binding affinity between the probe and target. The label can be covalently tethered to the probe or to the target, or can be an intercalating fluorophore in the reaction medium.

Abstract: The invention provides a homogeneous assay for nucleic acid hybridization. The fluorescent intensity of a hybridization medium containing a probe, a target and an intercalating agent is a function of the hybridization efficiency of the probe with respect to the target. The assay can detect specific hybridization between single-stranded probes and non-denatured double-stranded targets to form triplexes, thus obviating the need to denature the targets. The assay can also detect duplex hybridization complexes. The assay can be used to identify accessible regions in folded nucleotide sequences, to determine the number of mismatched pairs in a hybridization complex, and to map genomes.

Abstract: A complex includes: (1) a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; and (2) a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues, wherein: (a) at least one of the probe and the target is purified or synthetic; and (b) the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins. A method for assaying a target includes detecting formation of the complex.

Abstract: A method for assaying binding between a fluorophore-labeled compound and an unlabeled compound is provided. The method includes detecting a quenching effect on fluorescence emitted by the fluorophore-labeled compound resulting from binding. The binding is specific and other than nucleobase to nucleobase. The method is conducted without separating complexes of the fluorophore-labeled compound and the unlabeled compound from the fluorophore-labeled compound prior to quenching effect detecting, and without providing a signal quenching agent to quench fluorescent light. Preferably, the fluorophore-labeled compound is a nucleic acid and the unlabeled compound is a protein. The method can be used for a variety of applications, including screening for drug candidates having optimum binding properties, and quantifying the binding affinity of DNA binding proteins for nucleic acids.

Abstract: The invention provides homogeneous assay methods for nucleic acid hybridization, detection and evaluation. The assay includes obtaining signals from a test sample both before and during the application of a voltage to the test sample and correlating the signals, each of which is indicative of the binding affinity of the probe and the target to each other. The assay enables determining an extent of matching between the probe and the target, as the voltage can be calibrated so as to destabilize significantly any hybridization except perfectly complementary hybridization. The signals whose magnitude is correlated with binding affinity can be electrical conductance and/or fluorescent intensity. Preferably, both signal pairs are measured and compared so as to enhance the reliability of the assay. The assay can detect specific hybridization between single-stranded probes and non-denatured double-stranded targets to form triplexes, thus obviating the need to denature the targets.