Effect of Locked-Nucleic Acid on a Biologically Active G-Quadruplex. A Structure-Activity Relationship of the Thrombin Aptamer

Abstract

Abstract:Here we tested the ability to augment the biological activity of the thrombin aptamer, d(GGTTGGTGTGGTTGG), by using locked nucleic acid (LNA) to influence its G-quadruplex structure. Compared to un-substituted control aptamer, LNA-containing aptamers displayed varying degrees of thrombin inhibition. Aptamers with LNA substituted in either positions G5, T7, or G8 showed decreased thrombin inhibition, whereas LNA at position G2 displayed activity comparable to un-substituted control aptamer. Interestingly, the thermal stability of the substituted aptamers does not correlate to activity -the more stable aptamers with LNA in position G5, T7, or G8 showed the least thrombin inhibition, while a less stable aptamer with LNA at G2 was as active as the un-substituted aptamer. These results suggest that LNA substitution at sites G5, T7, and G8 directly perturbs aptamer-thrombin affinity. This further implies that for the thrombin aptamer, activity is not dictated solely by the stability of the G-quadruplex structure, but by specific interactions between the central TGT loop and thrombin and that LNA can be tolerated in a biologically active nucleic acid structure albeit in a position dependent fashion.

Anomeric DNA quadruplexes

“…It has been discussed in the literature that GQ thermal stability is not associated directly with the anticoagulant activity of a modified aptamer. 15 Our findings entirely agree with this conclusion. In this way, the most stable aptamer A3 (T m 60.3 °C) was shown to be inactive in clotting time tests.…”

“…studies on TBA analogs containing 3′-3′ and 5′-5′ junctions, LNA residues, internucleotide phosphorothioates, 2′-fluoroarabinose, isodeoxyguanosine and several other modified nucleotides have been reported. [11][12][13][14][15][16][17][18] Enhanced binding affinity to thrombin and improved anticoagulant and antithrombotic properties have been observed for certain TBA modifications. [11][12][13] In this study, we report the modification of TBA with anomeric α-nucleotides (Fig.…”

The effect of l-thymidine, acyclic thymine and 8-bromoguanine on the stability of model G-quadruplex structures

“…LNA derivatives, which are characterized by a restricted C3'-endo sugar pucker and syn conformation were also introduced in several positions of TBA. The results confirmed that LNA substitution in defined positions produced, syn-anti switch of glycosidic angle and, therefore, a mixture of alternative G-quadruplex structures were produced [10,11].…”

“…Nucleotides with special structural or physicochemical properties have been specifically substituted by natural nucleotides of TBA [3]. For example, locked nucleic acids (LNA) [10,11], unlocked nucleic acids (UNA) [12] or 2'-deoxy-2'-fluoro-β-D-arabinonucleic acid (2'F-ANA) [13] are derivatives with interesting backbone features that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 have been introduced in different positions of TBA. In addition, modification of nucleobases containing alkylating moieties or bases with different electronic density has been studied in order to understand the folding topology and stability of TBA [14].…”

Specific loop modifications of the thrombin‐binding aptamer trigger the formation of parallel structures

“…Significant changes in stability have also been found when thymidines in the lateral loops have been changed to other coding nucleotides [29,30], or to derivatives with modified backbones [31][32][33][34][35]. For example, the replacement of several thymidines at the loop positions with 2′-F-arabino derivatives always leads to a more stable G-quadruplex [31], whereas substitution by locked nucleic acids, unlocked nucleic acids and acyclic thymidines showed a position-dependent effect on TBA stability [32][33][34][35].…”