title: Analysis of in Situ LNA and DNA Hybridization Events on Microspheres.
abstract: The hybridization activity of single-stranded DNA and locked nucleic acid (LNA) sequences on microspheres is quantified in situ using flow cytometry. In contrast to conventional sample preparation for flow cytometry that involves several wash steps for posthybridization analysis, the current work entails directly monitoring hybridization events as they occur between oligonucleotide-functionalized microspheres and fluorescently tagged 9 or 15 base-long targets. We find that the extent of hybridization between single-stranded, immobilized probes and soluble targets generally increases with target sequence length or with the incorporation of LNA nucleotides in one or both oligonucleotide strands involved in duplex formation. The rate constants for duplex formation, on the other hand, remain nearly identical for all but one probe-target sequence combination. The exception to this trend involves the LNA probe and shortest perfectly matched DNA target, which exhibit a rate constant that is an order of magnitude lower than any other probe-target pair, including a mismatched duplex case. Separate studies entailing brief heat treatments to suspensions generally do not consistently yield appreciable differences in associated target densities to probe-functionalized microspheres.
authors.affiliation: School of Materials Science and Engineering, ‡Wallace H. Coulter Department of Biomedical Engineering, §Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , 771 Ferst Drive NW, Atlanta, Georgia 30332-0245, United States.
authors.forename: Ngozi A
authors.initials: NA
authors.lastname: Eze
authors.affiliation: School of Materials Science and Engineering, ‡Wallace H. Coulter Department of Biomedical Engineering, §Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , 771 Ferst Drive NW, Atlanta, Georgia 30332-0245, United States.
authors.forename: Richard S
authors.initials: RS
authors.lastname: Sullivan
authors.affiliation: School of Materials Science and Engineering, ‡Wallace H. Coulter Department of Biomedical Engineering, §Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , 771 Ferst Drive NW, Atlanta, Georgia 30332-0245, United States.
authors.forename: Valeria T
authors.identifier: 0000-0002-5303-7777
authors.initials: VT
authors.lastname: Milam
mesh_terms: D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded; D005434:Flow Cytometry; D061925:Immobilized Nucleic Acids / Q000737:chemistry; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D017422:Sequence Analysis, DNA
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D061925:Immobilized Nucleic Acids; D009711:Nucleotides; D009841:Oligonucleotides; C477371:locked nucleic acid