Real-time oligonucleotide hybridization kinetics monitored by resonant mirror technique

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dT GGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucle otide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was stud ied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association ( k(as)) and dissociation (k(dis)) rate constants at 25 degrees C: k(as) = 21 9 +/- 39 and 183 +/- 162 M-1 s(-1), k(dis) = (2.0 +/- 0.4) . 10(-3) and (4 +/- 1) . 10(-4) s(-1) for the duplexes (p14) . (ODN-11) and p14 . (ODN-14), respectively, The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), whic h forms a hairpin structure, does not associate with p14. The data were com pared with the results of melting curve detection and temperature-jump expe riments, The association rate constants for ODN-11 and ODN-14 are much slow er than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimate d in temperature-jump experiments.