Ac. Moses et A. Schepartz, KINETICS AND MECHANISM OF RNA-BINDING BY TRIPLEX TETHERED OLIGONUCLEOTIDE PROBES, Journal of the American Chemical Society, 119(48), 1997, pp. 11591-11597
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.