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 PD(I) and an internal complex PD(II) 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 (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M 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)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(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.
Biver
Tarita
T
Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Risorgimento, 35-56126 Pisa, Italy. tarita@dcci.unipi.it
Ciatto
Carlo
C
Secco
Fernando
F
Venturini
Marcella
M
D001665:Binding Sites; D003198:Computer Simulation; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D011370:Proflavine / Q000032:analysis* / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D012333:RNA, Messenger; D011370:Proflavine