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 degrees 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 (C-P/C-D). For C-P/C-D < 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) x 10(3) M-1), equilibrium constant for the growth step (K = (1.2 +/- 0.6) x 105 M-1), cooperativity parameter (q = 85) and rate constants for the growth step (k(r) = 1.2 x 10(7) M-1 s(-1), k(d) = 1.1 x 10(2) 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 C-P/C-D < 2.5. (c) 2006 Elsevier Inc. All rights reserved.