KINETICS AND MECHANISM OF RNA-BINDING BY TRIPLEX TETHERED OLIGONUCLEOTIDE PROBES

We have described a series of tethered oligonucleotide probes (tripler TOPs) that recognize one single-stranded and one double-stranded regi on of an RNA simultaneously through the formation of Watson-Crick and Hoogsteen base pairs, respectively. Here we describe studies on the ki netics and mechanism of tripler TOP . RREAU association and dissociati on. Because tripler TOP . RREAU complexes cannot be observed by direct electrophoretic methods, kinetics was monitored by use of a competiti ve electrophoretic mobility shift assay that quantified the effect of a tripler TOP on the association and dissociation rates of an electrop horetically stable TOP.RREAU complex. Association and dissociation rat e constants of tripler TOP.RREAU complexes were extracted from the exp erimental data by numerical integration. Tripler TOP.RREAU association reactions at 25 degrees C were characterized by rate constants betwee n (7.8 +/- 2.0) x 10(3) and (16 +/- 3) x 10(3) M-1 s(-1), while dissoc iation 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 as sociation of tripler TOP.RREAU complexes were insensitive to the lengt h and sequence of the 3'-oligonucleotide that mediates triple helix fo rmation. Rate constants fdr dissociation of tripler TOP.RREAU complexe s were sensitive to changes in tether length as well as the length and composition of the S'-oligonucleotide, Taken together, these data sug gest that tripler TOPs follow a kinetic pathway for binding RREAU whic h duplex formation is rate-limiting and precedes triple helix formatio n. The implication of our data with regard to the kinetics of triple h elix association within the context of a highly structured RNA is disc ussed.