Structure variations of TBA G-quadruplex induced by 2＋-O-methyl nucleotide in K＋ and Ca2＋ environments

Thrombin binding aptamer （TBA）, a 15-mer oligonucleo- tide of d（GGTTGGTGTGGTTGG） sequence, folds into a chair-type antiparallel G-quadruplex in the K＋ environ- ment, and each of two G-tetrads is characterized by a syn-anti-syn-anti glycosidic conformation arrangement. To explore its folding topology and structural stability, 2＇-0- methyl nucleotide （OMe） with the C31-endo sugar pucker conformation and anti glycosidic angle was used to selectively substitute for the guanine residues of G-tetrads of TBA, and these substituted TBAs were characterized using a circular dichroism spectrum, thermally differential spectrum, ultra- violet stability analysis, electrophoresis mobility shift assay, and thermodynamic analysis in K＋ and Ca2＋ environments. Results showed that single substitutions for syn-dG residues destabilized the G-quadruplex structure, while single substi- tutions for anti-dG residues could preserve the G-quadruplex in the K＋ environment. When one or two G-tetrads were modified with OMe, TBA became unstructured. In contrast, in Ca2＋ environment, the native TBA appeared to be un- structured. When two G-tetrads were substituted with OMe, TBA seemed to become a more stable parallel G-4 structure. Further thermodynamic data suggested that OMe-substitu- tions were an enthalpy-driven event. The results in this study enrich our understanding about the effects of nucleo- tide derivatives on the G-quadruplex structure stability in different ionic environments, which will help to design G-quadruplex for biological and medical applications.