Role of the third strand in the binding of proflavine and pt-proflavine to poly(rA).2poly(rU): a thermodynamic and kinetic study.

The interactions of triple strands of poly(rA).2poly(rU) with proflavine (PR) and the proflavine cis-platinum derivative [{PtCl (tmen)} 2{NC 13H 7(NCH 2CH 2) 2}] (+) (PRPt) are examined at pH 7.0, T = 25 degrees C, and 0.2 M ionic strength by spectrophotometry, spectrofluorometry, circular dichroism, viscosimetry, stopped-flow, and T-jump relaxation techniques. The melting experiments demonstrate that both drugs tend to destabilize the triplex structure, although the PRPt effect is more relevant. By contrast, both drugs tend to slightly stabilize the duplex structure. The viscosity and circular dichroism measurements show that, at a low dye-to-polymer ratio ( C D/ C P), the binding is intercalative, whereas at high C D/ C P values, the external binding dominates. The binding kinetics and equilibria have been investigated over the C D/ C P region, where intercalation is operative. Both drugs bind to the RNA triplex according to the excluded site model. With PR, two kinetic effects have been observed, whereas with PRPt, only one has been observed. The results are interpreted according to the reaction schemes D + S right arrow over left arrow DS I, with PRPt, and D + S right arrow over left arrow DS I right arrow over left arrow DS II, with PR. The electrostatic contribution to the formation activation energy for DS I is similar (40%) for both systems. The results suggest that DS I is a partially intercalated species. Absence of the second step with PRPt is put down to groove interaction of the Pt-containing moiety, which prevents the PR residue from further penetration through the base pairs to form the fully intercalated complex, DS II. Comparison with the binding of the same drugs to the duplex reveals that the occupation of the major groove in poly(rA).2poly(rU) by the third strand plays a critical role in the kinetic behavior.

Potentiometric characterization of ethidium bromide and of its reactions with nucleic acids.

A liquid membrane electrode that allows the concentration of ethidium ion (Ed(+)) to be measured selectively and accurately in the range of 0.1 microM to 5 mM is made. For Ed(+) concentrations less than 1 microM or more than 0.1 mM, the trend is no longer linear, and the causes of this behavior are discussed. The mean activity coefficient of ethidium bromide exhibits deviations from the Debye-Huckel limiting law that are interpreted in terms of aggregate formation. The stability constants for Ed(2)(2+) and Ed(2)Br(+) are 230 kg mol(-1) and 3.0 x 10(4) kg(2) mol(-2), respectively. In NaCl solutions, clusters involving up to 4 Ed(+) units are detected and their stability constants are evaluated. The intercalation of ethidium into poly(A).poly(U) in 1M NaCl is investigated by the above electrode, and the results are compared with those obtained by spectrophotometry. The data are analyzed in terms of Scatchard plots. The potentiometric method is more accurate than the spectrophotometric one at low values of the binding degree (r) where negative deviations from linearity are observed. The deviations are ascribed to a cooperative behavior rather than to artifacts caused by minor systematic errors.