Investigating the kinetics of DNA-DNA and PNA-DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy.
Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin-streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.
Kambhampati
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D
Max-Planck-Institut für Polymerforschung, Postfach 3148, Ackermannweg 10, 55128 Mainz, Germany.
Nielsen
P E
PE
Knoll
W
W
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D015342:DNA Probes; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
D015342:DNA Probes; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D004247:DNA