title: Monitoring molecular beacon DNA probe hybridization at the single-molecule level.
abstract: We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.
authors.affiliation: Center for Research at the Interface of Bio/nano, Department of Chemistry and the McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Yao
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Fang
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yokota
authors.forename: Toshio
authors.initials: T
authors.lastname: Yanagida
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
mesh_terms: D015374:Biosensing Techniques; D015342:DNA Probes / Q000737:chemistry*; D018076:DNA, Complementary / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization / Q000379:methods*
chemical_list: D015342:DNA Probes; D018076:DNA, Complementary; D005456:Fluorescent Dyes