A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

A thrombin binding aptamer (TBA) is a synthetic 15-nt DNA oligomer that binds human alpha thrombin with high specificity and reversibly suspends the coagulation cascade and platelet formation. It has been established that TBA folds intramolecularly into a G-quadruplex (GQ) structure in solution, forming two G tetrads and three loops1. Two TT loops play a critical role in recognizing the target protein at its fibrinogen binding site (exosite I). The most recent high-resolution crystallographic studies of the TBA-thrombin complex have provided details of the interaction between thrombin and TBA loops2,3. It was shown that, in TT loops, T4 and T13 form polar interactions with the amino acid residues of exosite I, while T3 and T12 form hydrophobic contacts. The role of the central TGT loop has been arguably attributed to the binding of the aptamer to the heparin binding site of thrombin (exosite II)4–6. However, a recent crystallographic study suggests that the TGT loop is unlikely to be involved in thrombin binding and interacts with neither the fibrinogen nor the heparin binding sites of the protein3. Although located away from the binding site of the aptamer, the central loop is a key element of TBA structure. Numerous studies on modifications of TBA with non-natural nucleotides indicate that the improved anticoagulant properties or higher binding affinities of modified variants are often associated with the transformation of the TGT loop. In this way, a few advanced TBA analogs containing 2′ -deoxyisoguanosine, 2′ -deoxy-2′ -fluoroarabinonucleotides, 4-thio-2′ -deoxyuridine, 5-hydroxymethyl -2′ -deoxyuridine, D-/L-isothymidine and UNA (unlocked nucleic acid) residues in the TGT loop have been described7–12. Artificial residues that have been used for numerous TBA modifications feature altered sugar units, nucleobases, and an internucleotide backbone13. Hydrophobic interactions in TBA studies have mainly been considered in the context of binding intercalators and fluorophores14–18. Replacement of G nucleobases within TBA G-tetrads with fluorescent 8-aryl-dG residues has recently been reported19. TBA analogs containing universal bases have not been described. 5-Nitroindole (NI) has long been known as the best universal base analogue for double stranded DNA20,21. This non-natural nucleoside has been