Matching base-pair number dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy.
Two single-stranded DNA oligonucleotides consisting of complementary base-pairs can form double strands. This phenomenon is well studied in solutions, however, in order to clarify the physical mechanism of the hybridization occurring at a solid/solution interface, we studied the kinetics by surface plasmon fluorescence spectroscopy (SPFS): one single-stranded oligo-DNA (probe-DNA) was immobilized on the substrate, the other one (target-DNA) labelled with a fluorescent probe was added to the flow cell. After hybridization, the chromophores could be excited by the surface plasmon mode and their fluorescence detected with high sensitivity. The dependence of the k(on) and k(off) rate constants on the length of the hybridizing oligonucleotides was investigated by using a MM0 series (no mismatch) and the kinetics was found to be well described by a Langmuir adsorption model. From these measurements we found that also in the case of surface hybridization the affinity of the duplexes decreases as the number of matching base-pairs decreases from 15 to 10. In order to show that SPFS is the powerful technique with high sensitivity, the hybridization process for mixed target-oligos was measured by SPFS and analyzed by an expanded Langmuir model in which two components of target-oligo can bind to probe-DNA at the sensor surface competitively. Two sets of the k(on) and k(off) obtained from the experiment are successfully consistent with the k(on) and k(off) obtained from experiments for single (pure) target-DNA.
Tawa
Keiko
K
Max-Planck-Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany. tawa-keiko@aist.go.jp
Yao
Danfeng
D
Knoll
Wolfgang
W
D020029:Base Pairing; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation* / Q000379:methods; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D020349:Surface Plasmon Resonance / Q000295:instrumentation* / Q000379:methods
D004247:DNA