Counterion association with native and denatured nucleic acids: An experimental approach

The melting temperature of the poly(dA) . poly(dT) double helix is exquisit ely sensitive to salt concentration, and the helix-to-coil transition is sh arp. Modem calorimetric instrumentation allows this transition to be detect ed and characterized with high precision at extremely low duplex concentrat ions. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influenc e of other solutes on solution properties. We demonstrate how the temperatu re associated with poly(dA).poly(dT) melting can be used to define the chan ge in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We u se this information to critically evaluate features of counterion condensat ion theory, as well as to illustrate "crosstalk" between different, non-con tacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA).poly(dT), in the presence of excess genomic salmon s perm DNA, or in the presence of one of two synthetic RNA polymers (the poly (rA).poly(rU) duplex or the poly(rU).poly(rA).poly(rU) triplex). We find th at these additions cause a shift in the melting temperature of poly(dA).pol y(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed t(m) shift does not depend significantly on whether the added pol ymer is RNA or DNA, but it does depend on the number of strands making up t he helix of the added polymer. We ascribe the observed changes in melting t emperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and i ts associated counterions. We refer to this communication between non-conta cting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of t(m) versus log[Na+] for poly(dA).poly(dT), we estimate the magnitude of the apparent change in ionic strength resultin g from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical consideratio ns correctly predict the direction of the t(m) shift (the melting temperatu re increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5 % of the concentration o f the added duplex DNA or RNA (in mol phosphate), and an additional apparen t increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent incre ase of 14.5 %. For the poly(rU) . poly(rA).poly(rU) triplex, the total appa rent increase in ionic strength corresponds to about 13.6 % of the amount o f added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation c oncentration induced by the presence and/or the transition of one of the so lute molecules. We note that our results are general, so one can use a diff erent solute probe sensitive to proton binding to characterize subtle chang es in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probin g counterion environments, and in terms of potential regulatory mechanisms. (C) 2001 Academic Press.