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Reduction in Dynamics of Base pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer.

ABSTRACT: We have used magnetization transfer NMR experiments to measure the exchange rate constant (k_ex) of the imino protons in the unbound, cocaine-bound, and quinine-bound forms of the cocaine-binding DNA aptamer. Both long-stem 1 (MN4) and short-stem 1 (MN19) variants were analyzed, corresponding to structures with a prefolded secondary structure and ligand-induced-folding versions of this aptamer, respectively. The k_ex values were measured as a function of temperature from 5 to 45°C to determine the thermodynamics of the base pair opening for MN4. We find that the base pairs close to the ligand-binding site become stronger upon ligand binding, whereas those located away from the binding site do not strengthen. With the buffer conditions used in this study, we observe imino ¹H signals in MN19 not previously seen, which leads us to conclude that in the free form, both stem 2 and parts of stem 3 are formed and that the base pairs in stem 1 become structured or more rigid upon binding. This is consistent with the k_ex values for MN19 decreasing in both stem 1 and at the ligand-binding site. Based on the temperature dependence of the k_ex values, we find that MN19 is more dynamic than MN4 in the free and both ligand-bound forms. For MN4, ligand-binding results in the reduction of dynamics that are localized to the binding site. These results demonstrate that an aptamer in which the base pairs are preformed also experiences a reduction in dynamics with ligand binding.

SUBMITTER: Churcher ZR

PROVIDER: S-EPMC7499103 | biostudies-literature |

REPOSITORIES: biostudies-literature

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