FIGURE 3.
Influence of incubation temperature and time on the as548 oligonucleotide-induced dimerization of 1–561 RNA. The 1–444 RNA (A), 1–561 RNA with the as548 oligonucleotide (B), or 1–561 RNA without oligonucleotide (C) were incubated in dimer buffer for 30 min at the indicated temperatures and subjected to electrophoresis on TBE agarose gel run at 28°C. In B, the 1–561 RNA was incubated with a 20-fold excess of the antisense DNA oligonucleotide as548, which is complementary to nt 528–548. The band below the indicated monomer band in A and the two faint bands between monomer and dimer bands in B probably represent low-abundance conformers of monomer (in A) or dimer (in B) species. Control lanes C are described in the previous figure. (D) The time-dependent dimerization of 1–561 RNA at 55°C in the presence of the as548 oligonucleotide was monitored on a TBE agarose gel at 28°C. (Lanes 1–16) Incubation times of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 min, respectively. (E) Plots of the kinetic data for 1–526 RNA and 1–561 RNA with or without as548 (represented by closed circles, open squares, and open diamonds, respectively). [M]t is the concentration of monomer at time t, and [M]0 is the initial concentration of dimerization-competent monomer.