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Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method.

ABSTRACT: A variation of affinity capillary electrophoresis, called the replacement ion (RI) method, has been developed to measure the binding of monovalent cations to random sequence, double-stranded (ds) DNA. In this method, the ionic strength is kept constant by gradually replacing a non-binding ion in the solution with a binding ion and measuring the mobility of binding and non-binding analytes as a function of binding ion concentration. The method was validated by measuring the binding of Li+ ions to adenosine nucleotides; the apparent dissociation constants obtained by the RI method are comparable to literature values obtained by other methods. The binding of Tris+, NH4+, Li+, Na+, and K+ to dsDNA was then investigated. The apparent dissociation constants observed for counterion binding to a random-sequence 26-base pair (bp) oligomer ranged from 71 mM for Tris+ to 173 mM for Na+ and K+. Hence, positively charged Tris buffer ions will compete with other monovalent cations in Tris-buffered solutions. The bound cations identified in this study may correspond to the strongly correlated, tightly bound ions recently postulated to exist as a class of ions near the surface of dsDNA (Tan, Z.-J., and Chen, S.-J. (2006) Biophys. J. 91, 518-536). Monovalent cation binding to random-sequence dsDNA would be expected to occur in addition to any site-specific binding of cations to A-tracts or other DNA sequence motifs. Single-stranded DNA oligomers do not bind the five tested cations under the conditions investigated here.

SUBMITTER: Stellwagen E

PROVIDER: S-EPMC2532990 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method.

Stellwagen Earle E Dong Qian Q Stellwagen Nancy C NC

Biochemistry 20070125 7

A variation of affinity capillary electrophoresis, called the replacement ion (RI) method, has been developed to measure the binding of monovalent cations to random sequence, double-stranded (ds) DNA. In this method, the ionic strength is kept constant by gradually replacing a non-binding ion in the solution with a binding ion and measuring the mobility of binding and non-binding analytes as a function of binding ion concentration. The method was validated by measuring the binding of Li+ ions to ...[more]

PMID: 17253778

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Project description:A kissing loop is a highly stable complex formed by loop-loop base-pairing between two RNA hairpins. This common structural motif is utilized in a wide variety of RNA-mediated processes, including antisense recognition, substrate recognition in riboswitches, and viral replication. Recent work has shown that the Tar-Tar(*) complex, an archetypal kissing loop, can form without Mg(2+), so long as high concentrations of alkali chloride salts are present. Interestingly, the stability of the complex is found to decrease with increasing cation size. In this work, we used molecular simulations to develop a molecular-level understanding of the origins of the observed counterion specificity. The ionic atmosphere of the Tar-Tar(*) complex was examined in the presence of 800 mm (where m denotes molality) NaCl, KCl, or CsCl. We used spatial free-energy density profiles to analyze differences in counterion accumulation at different spatial extents from the RNA molecule. We found that the lowest free-energy levels, which are situated in the vicinity of the loop-loop interface, can accommodate roughly two counterions, irrespective of counterion type. However, as we moved into higher free-energy levels, which are farther from the loop-loop interface, we observed increased differences in the numbers of accumulated counterions, with this number being largest for Na(+) and smallest for Cs(+). We analyzed the source of these differences and were able to attribute these to two distinct features: The extent of partial dehydration varies based on cation type; the smaller the cation, the greater the degree of dehydration. While smaller ions bind their first-hydration-shell water molecules more tightly than larger ions, they are also able to shed these water molecules for stronger electrostatic interactions with the RNA molecule. Secondly, we observed a distinct asymmetry in the numbers of accumulated cations around each hairpin in the Tar-Tar(*) complex. We were able to ascribe this asymmetry to the presence of a guanine tract in the Tar hairpin, which facilitates partial dehydration of the counterions. However, the smaller ions compensate for this asymmetry by forming a belt around the loop-loop interface in intermediate free-energy levels. As a result, the degree of asymmetry in counterion accumulation around individual hairpins shows an inverse correlation with the experimentally observed cation specificity for the stability of Tar-Tar(*) (i.e., the smaller the asymmetry, the greater the experimentally observed stability). This in turn provides a plausible explanation for why the smaller cations help stabilize the Tar-Tar(*) complex better than the larger cations. These findings suggest that the specific sequence and structural features of the Tar-Tar(*) complex may be the source of the experimentally observed cation specificity in Tar-Tar(*) stability. Our results lead to testable predictions for how changes in sequence might alter the observed counterion specificity in kissing loop stability.

Dataset Information

Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method.

Publications

Quantitative analysis of monovalent counterion binding to random-sequence, double-stranded DNA using the replacement ion method.

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