title: DNA meter: Energy tunable, quantitative hybridization assay.
abstract: We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial "pre-binding" of a probe strand modulates its effective "availability" for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a "tuning fork," since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such "tuning forks," one can construct C*P "energy ladders" capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a "DNA Meter." Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity.
authors.affiliation: Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Rd., Piscataway, NJ, 08854; Rational Affinity Devices, LLC.
authors.forename: William
authors.initials: W
authors.lastname: Braunlin
authors.forename: Jens
authors.initials: J
authors.lastname: Völker
authors.forename: G Eric
authors.initials: GE
authors.lastname: Plum
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Breslauer
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D006801:Humans; D009693:Nucleic Acid Hybridization
chemical_list: D004247:DNA