Dye-induced aggregation of single stranded RNA: A mechanistic approach

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 °C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PDI and an internal complex PDII form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (CP/CD). For CP/CD < 2.5, cooperative formation of stacks external to polymer strands prevails (PDI). Equilibria and T-jump experiments, performed at I = 0.1 M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g = 1), equilibrium constant for the nucleation step (K∗ = (1.4 ± 0.6) × 103 M−1), equilibrium constant for the growth step (K = (1.2 ± 0.6) × 105 M−1), cooperativity parameter (q = 85) and rate constants for the growth step (kr = 1.2 × 107 M−1 s−1, kd = 1.1 × 102 s−1). Stopped-flow experiments, performed at low ionic strength (I = 0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that CP/CD < 2.5.