We report a general method for screening, in solution, the impact of deviations from canonical WatsonCrick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how f luorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed ''reference'' duplex (usually a Watson-Crick duplex) and a related ''test'' duplex containing a lesion͞alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a f luorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized f luorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of f luorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.Thermodynamic studies of nucleic acid duplexes containing base adducts, mismatches, bulges, and other deviations from canonical Watson-Crick pairing͞stacking reveal that relatively modest structural defects can be accompanied by profound energetic consequences (1-3). In fact, it has been shown that such defects͞lesions can result in significant destablizations of the duplex, with magnitudes that are well beyond that which can be rationalized in terms of local structural perturbations (3). Because the free energy term, ⌬G o , quantifies duplex stability, a systematic detection͞screening for and comparison of defect-induced duplex destabilization requires a method (preferably a high throughput one) that yields accurate ⌬G o determinations.The free energy change associated with formation of a duplex can be obtained from the equilibrium association constant, K, via the well known relation ⌬G o ϭ ϪRTlnK. Because the values of K for oligonucleotide duplexes typically are beyond the range accessible by standard titration techniques, most thermodynamic studies of DNA have relied on indirect methods for determination of K and ⌬G o (4). These methods often m...
The macrophage scavenger receptor (MSR), involved in the uptake of oxidized LDL, binds a variety of polyanionic ligands, and in particular shows selectivity for tetraplex forms of nucleic acids. The ligand binding region has been shown to lie in the triple-helical collagen-like domain of MSR. A model peptide-nucleic acid system is presented here to clarify how the triple-helical motif of MSR recognizes and binds tetraplex nucleic acids. The triple-helical peptide MSR-1, with the sequence (POG)3PKGQKGEKG(POG)4, contains a nine amino acid basic sequence implicated in MSR ligand binding, flanked by Pro-Hyp-Gly triplets to provide stability. The ability of this triple-helical MSR-1 peptide to bind to and perturb the conformation of nucleic acids in tetraplex, duplex, and single-stranded states was assessed by monitoring changes in the nucleic acid circular dichroism spectrum in the 240-300 nm region. Our results show that the triple-helical MSR-1 peptide binds to tetraplex poly(I) in a stoichiometric manner and is capable of reproducing the discrimination exhibited by the native MSR molecule for tetraplex over double-stranded or single-stranded nucleic acid states. The triple-helical reference peptide (POG)10 does not bind to tetraplex poly(I), suggesting that binding requires the highly basic 9-mer sequence from MSR that is included in MSR-1. The MSR-1 peptide did not perturb the CD spectra of a series of other tetraplex nucleic acids, indicating that it does not model the broader specificity that MSR shows under physiological conditions. Models of possible interactions between a triple-helical peptide and a tetraplex polynucleotide are proposed on the basis of the stoichiometry observed for the complex between triple-helical MSR-1 and tetraplex poly(I).
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