title: High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.
abstract: Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.
authors.affiliation: Department of Chemical Engineering and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890, United States.
authors.forename: Johnathan M
authors.initials: JM
authors.lastname: Goldman
authors.forename: Li Ang
authors.initials: LA
authors.lastname: Zhang
authors.forename: Arunava
authors.initials: A
authors.lastname: Manna
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Armitage
authors.forename: Danith H
authors.initials: DH
authors.lastname: Ly
authors.forename: James W
authors.initials: JW
authors.lastname: Schneider
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D035683:MicroRNAs / Q000032:analysis*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; D009696:Nucleic Acids; D020135:Peptide Nucleic Acids; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; D004247:DNA