Abstract: Heteropolymeric triplexes and quadruplexes and methods for making them; the use of accelerator agents such as cations to create them; the use of fluorescent intercalators and fluorescent probe-bound non-intercalators to detect them.

Abstract: A purification method includes bonding a probe to a target in a sample to form a complex, and separating the sample from the complex to separate in a sequence specific manner the target from the sample. The complex, which is immobilized on a support, is a duplex, triplex or quadruplex formed 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 kit for performing the method includes the support and the probe.

Abstract: An aptamer contains at least two parallel or antiparallel heteropolymeric nucleobase-containing sequences bonded together by Watson-Crick complementary base interaction or by homologous base interaction, provided that: (a) when the aptamer is single-stranded, the at least two sequences are bonded together by homologous base interaction; and (b) when the aptamer is a duplex and the at least two sequences are antiparallel to each other, the at least two sequences are bonded together by homologous base interaction. The aptamer can be used to bind ligands or to catalyze reactions when functioning as an aptazyme.

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: A method for homogeneously assaying biopolymer bonding includes obtaining signals from a test sample before, during and/or after the application of stimulus to the test sample and correlating the signals. The signals, whose magnitude correlate with binding affinity, can be, for example, electrical conductance and/or fluorescent intensity. The stimulus can be, for example, electric voltage and/or laser radiation. Preferably, different types of signals are measured and compared so as to enhance the reliability of the assay.

Abstract: An improved method of forming a specific complex between a probe containing probe nucleobases and a target containing target nucleobases, includes mixing the probe and the target under hybridizing conditions, wherein the probe and/or the target is conjugated to a blocking agent, which enhances the avidity and/or specificity of hybridization, whether by Watson-Crick motif or by homologous binding motif. The blocking agent contains at least one nucleobase and can be, e.g., a free nucleobase, a nucleoside or a nucleotide.

Abstract: An apparatus for assaying specific binding of a probe to a target, includes: a sample support; a light source; an optical train; a light detector; an electricity source; an electrical property detector; and a data analysis device adapted to: (a) compare an optical determination of binding with an electrical determination of binding, or (b) compare a pre-electrification determination of binding with a post-electrification determination of binding.

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.