Search

Metal-mediated base pairs expand the repertoire of nucleic acid structures and dynamics. Here we report solution structures and dynamics of duplex DNA containing two all-natural C-Hg II -T metallo base pairs separated by six canonical base pairs. NMR experiments reveal a 3:1 ratio of well-resolved structures in dynamic equilibrium. The major species contains two (N3)T-Hg II -(N3)C base pairs in a predominantly B-form helix. The minor species contains (N3)T-Hg II -(N4)C base pairs and greater A-form characteristics. Ten-fold different 1 J coupling constants ( 15 N, 199 Hg) are observed for (N3)C-Hg II (114 Hz) versus (N4)C-Hg II (1052 Hz) connectivities, reflecting differences in cytosine ionization and metal-bonding strengths. Dynamic interconversion between the two types of C-Hg II -T base pairs are coupled to a global conformational exchange between the helices. These observations inspired the design of a repetitive DNA sequence capable of undergoing a global B-to-A-form helical transition upon adding Hg II , demonstrating that C-Hg II -T has unique switching potential in DNA-based materials and devices.

Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T-T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6-19°C), as compared to those containing C-T mismatches (ΔTm = 1-4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C-HgII-T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs - even in situations where metal binding has little or no impact on the thermal stability of the duplex.

A fluorescent surrogate of thymidine called DMAT was used for the first fluorescence-based study of HgII binding to discrete T-T sites in duplex DNA. The fluorescent properties of DMAT-A base pairs were highly sensitive to wild-type T-HgII-T base pair formation at an adjacent site, allowing for a determination of the precise thermodynamic and kinetic parameters of these metal binding reactions. T-HgII-T complexes exhibited equilibrium dissociation constants of Kd ≈ 8-50 nM. These high-affinity binding interactions are characterized by very slow association and dissociation kinetics (kon ≈ 104- 105 M-1s-1, koff ≈ 10-4 - 10-3s-1), revealing exceptionally high kinetic stabilities of T-HgII-T base pairs (half-lives = 0.3-1.3 h). Duplex DNA containing DMAT and no T-T mismatch exhibited nonspecific HgII binding affinities of Kd ≈ 2.0 μM. The high kinetic stabilities of T-HgII-T resulted in the inhibition of dynamic processes such as DNA strand invasion and strand displacement during enzymatic DNA synthesis, which led to premature chain termination. These results demonstrated that T-HgII-T base pairs are kinetically distinct from T-A base pairs and therefore are likely to disrupt DNA metabolism in vivo.

The thymidine analogue DMA T was used for the first fluorescence-based study of direct, site-specific metal binding reactions involving unmodified nucleobases in duplex DNA. The fluorescence properties of DMA T-A base pairs were highly sensitive to mercury binding reactions at T-T mismatches located at an adjacent site or one base pair away. This allowed for precise determination of the local kinetic and thermodynamic parameters of T-Hg II -T binding reactions. The on- and off-rates of Hg II were surprisingly slow, with association rate constants (k on ) ≈ 10 4 -10 5 M -1 s -1 , and dissociation rate constants (k off ) ≈ 10 -4 -10 -3 s -1 ; giving equilibrium dissociation constants (K d ) = 8-50 nM. In contrast, duplexes lacking a T-T mismatch exhibited local, nonspecific Hg II binding affinities in the range of K d = 0.2-2.0 μM, depending on the buffer conditions. The exceptionally high kinetic stabilities of T-Hg II -T metallo-base pairs (half-lives = 0.3-1.3 h) perturbed dynamic processes including DNA strand displacement and primer extension by DNA polymerases that resulted in premature chain termination of DNA synthesis. In addition to providing the first detailed kinetic and thermodynamic parameters of site-specific T-Hg II -T binding reactions in duplex DNA, these results demonstrate that T-Hg II -T base pairs have a high potential to disrupt DNA metabolism in vivo.