Stabilization of DNA duplex by 2-substituted adenine as a minor groove modifier
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Duplex (building)
Deoxyadenosine
Abstract The DNA double helix structure is stabilized by base-pairing and base-stacking interactions. However, a comprehensive understanding of dinucleotide base-stacking energetics is lacking. Here we combined multiplexed DNA-based point accumulation in nanoscale topography (DNA-PAINT) imaging with designer DNA nanostructures and measured the free energy of dinucleotide base stacking at the single-molecule level. Multiplexed imaging enabled us to extract the binding kinetics of an imager strand with and without additional dinucleotide stacking interactions. The DNA-PAINT data showed that a single additional dinucleotide base stacking results in up to 250-fold stabilization for the DNA duplex nanostructure. We found that the dinucleotide base-stacking energies vary from −0.95 ± 0.12 kcal mol −1 to −3.22 ± 0.04 kcal mol −1 for C|T and A|C base-stackings, respectively. We demonstrate the application of base-stacking energetics in designing DNA-PAINT probes for multiplexed super-resolution imaging, and efficient assembly of higher-order DNA nanostructures. Our results will aid in designing functional DNA nanostructures, and DNA and RNA aptamers, and facilitate better predictions of the local DNA structure.
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Aptamer
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Groove (engineering)
Sequence (biology)
Molecular model
Base (topology)
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The thermal stabilities and structures of B−Z junction forming DNA duplexes possessing A/C or G/T base pair mismatches were compared to those of corresponding duplexes possessing perfect matched base pairs. The upper strands of the duplexes have a generalized sequence 5'-(5meCG)-LMN-GACTG-3', where L stands for A or G while M and N are permutations of pyrimidines. The lower strands were either complementary or were such as to create an A/C or G/T mismatch at the position corresponding to L, M, or N. Optical melting and circular dichroism studies were used to investigate the thermal stabilities and structures of both the mismatched base pair and the perfect matched base pair duplexes. Incorporating mismatched A/C or G/T base pairs did not noticeably affect the conformations of the duplexes in 115 mM Na+ but resulted in perturbed B−Z conformations at 4.5 M Na+. For any mismatched base pair duplex, the B-DNA domain of the hybrid B−Z structure formed at 4.5 M Na+ is significantly perturbed while the Z-DNA domain is less perturbed by the presence of the mismatched base pairs. The presence of a mismatch destabilizes a duplex relative to the perfect matched base pair duplex by 1.7−10.0 kcal/mol depending upon position of the mismatch, type of mismatch base pair involved, and Na+ concentration. The thermodynamic destabilization of a mismatched base pair duplex relative to the perfect matched base pair duplex arises from perturbations in nearest neighbor interactions and hydrogen bonding. In general, we observed that the incorporation of an A/C or G/T base pair mismatch in place of a perfect matched base pair at or near a B−Z junction results in a relatively large change in enthalpy and entropy to produce a significant change in the free energy of the duplex to single strand transition. At 4.5 M Na+, where the duplexes possess perturbed B−Z junctions, the farther away from the junction that the mismatch is, the greater the extent of the destabilization.
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Thermal Stability
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Stable and selective DNA base pairing by metal coordination was recently demonstrated with nucleotides containing complementary pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) bases (Meggers, E.; Holland, P. L.; Tolman; W. B.; Romesberg, F. E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714−10715). To understand the structural consequences of introducing this novel base pair into DNA we have solved the crystal structure of a duplex containing the metallo-base pair. The structure shows that the bases pair as designed, but in a Z-DNA conformation. The structure also provides a structural explanation for the B- to Z-DNA transition in this duplex. Further solution studies demonstrate that the metallo-base pair is compatible with Z- or B-DNA conformations, depending on the duplex sequence.
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We study the thermal denaturation of double-stranded DNA, i.e., separation of its two strands upon heating. A simple homo-polymer model is used to account for the effect of base stacking on the thermal stability of DNA. We find that stacking influences the stability in a nontrivial way: It not only enhances the stability but also makes the denaturation transition sharp. While stacking between bound monomers stabilizes DNA as does base pairing, stacking in unbound parts (or loops) rather destabilizes DNA--the overall stability is, however, enhanced by stacking.
Thermal Stability
Denaturation (fissile materials)
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Nucleic Acid Denaturation
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ABSTRACT DNA double helix structure is stabilized by the base-pairing and the base-stacking interactions. Base-stacking interactions originating from hydrophobic interactions between the nucleobases predominantly contribute to the duplex stability. A comprehensive understanding of dinucleotide base-stacking interactions is lacking owing to the unavailability of sensitive techniques that can measure these weak interactions. Earlier studies attempting to address this question only managed to estimate the base-pair stacking interactions, however, disentangling individual base-stacking interactions was enigmatic. By combining multiplexed DNA-PAINT imaging with designer DNA nanostructures, we experimentally measure the free energy of dinucleotide base-stacking at the single-molecule level. Multiplexed imaging enabled us to extract binding kinetics of an imager strand with and without additional dinucleotide stacking interactions in a single imaging experiment, abolishing any effects of experimental variations. The DNA-PAINT data showed that a single additional dinucleotide base-stacking results in as much as 250-fold stabilization of the imager strand binding. We found that the dinucleotide base-stacking energies vary from -1.18 ± 0.17 kcal/mol to -3.57 ± 0.08 kcal/mol for C|T and A|C base-stackings, respectively. We demonstrate the application of base-stacking energetics in designing DNA-PAINT probes for multiplexed super-resolution imaging. Our results will aid in designing functional DNA nanostructures, DNA and RNA aptamers, and facilitate better predictions of the local DNA structure.
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We investigated molecular crowding effects on the thermodynamic stability of Hoogsteen and Watson-Crick base pairs in an intramolecular duplex and triplex. The melting temperature (Tm) of Hoogsteen base pair formations in the triplex and the duplex increased 3.7 degrees C and 3.2 degrees C, respectively, by adding 20 wt% PEG 200. On the other hand, the Tm of Watson-Crick base pair formations in the triplex and the duplex decreased 5.7 degrees C and 5.2 degrees C, respectively. These results suggested that molecular crowding conditions generally stabilized and destabilized Hoogsteen and Watson-Crick base pairs, respectively, even in the different DNA structures.
Duplex (building)
Nucleic Acid Denaturation
Chemical Stability
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Recently, we reported the first artificial nucleoside for alternative DNA base pairing through metal complexation (J. Org. Chem. 1999, 64, 5002−5003). In this regard, we report here the synthesis of a hydroxypyridone-bearing nucleoside and the incorporation of a neutral Cu2+-mediated base pair of hydroxypyridone nucleobases (H−Cu−H) in a DNA duplex. When the hydroxypyridone bases are incorporated into the middle of a 15 nucleotide duplex, the duplex displays high thermal stabilization in the presence of equimolar Cu2+ ions in comparison with a duplex containing an A−T pair in place of the H−H pair. Monitoring temperature dependence of UV-absorption changes verified that a Cu2+-mediated base pair is stoichiometrically formed inside the duplex and dissociates upon thermal denaturation at elevated temperature. In addition, EPR and CD studies suggested that the radical site of a Cu2+ center is formed within the right-handed double-strand structure of the oligonucleotide. The present strategy could be developed for controlled and periodic spacing of neutral metallobase pairs along the helix axis of DNA.
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Duplex (building)
Helix (gastropod)
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Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na + . From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
Helix (gastropod)
Duplex (building)
Nucleic Acid Denaturation
Thermal Stability
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