We have described a series of tethered oligonucleotide probes (tripler TOPs) that recognize one single-stranded and one double-stranded region of an RNA simultaneously through the formation of Watson-Crick and Hoogsteen base pairs, respectively. Here we describe studies on the kinetics and mechanism of tripler TOP . RREAU association and dissociation. Because tripler TOP . RREAU complexes cannot be observed by direct electrophoretic methods, kinetics was monitored by use of a competitive electrophoretic mobility shift assay that quantified the effect of a tripler TOP on the association and dissociation rates of an electrophoretically stable TOP.RREAU complex. Association and dissociation rate constants of tripler TOP.RREAU complexes were extracted from the experimental data by numerical integration. Tripler TOP.RREAU association reactions at 25 degrees C were characterized by rate constants between (7.8 +/- 2.0) x 10(3) and (16 +/- 3) x 10(3) M-1 s(-1), while dissociation reactions were characterized by rate constants between (3.3 +/- 1.0) x 10(-4) and (5.4 +/- 2.0) x 10(-2) s(-1). Rate constants for association of tripler TOP.RREAU complexes were insensitive to the length and sequence of the 3'-oligonucleotide that mediates triple helix formation. Rate constants fdr dissociation of tripler TOP.RREAU complexes were sensitive to changes in tether length as well as the length and composition of the S'-oligonucleotide, Taken together, these data suggest that tripler TOPs follow a kinetic pathway for binding RREAU which duplex formation is rate-limiting and precedes triple helix formation. The implication of our data with regard to the kinetics of triple helix association within the context of a highly structured RNA is discussed.