Aptamer-peptide conjugates as a new strategy to modulate human α-thrombin binding affinity

Aptamers are single-stranded RNA or DNA molecules that specifically recognize their targets and have proven valuable for functionalizing sensitive biosensors. α-thrombin is a trypsin-like serine proteinase which plays a crucial role in haemostasis and thrombosis. An abnormal activity or overexpression of this protein is associated with a variety of diseases. A great deal of attention was devoted to the construction of high-throughput biosensors for accurately detect thrombin for the early diagnosis and treatment of related diseases. Herein, we propose a new approach to modulate the interaction between α-thrombin and the aptamer TBA15. To this end, TBA15 was chemically conjugated to two peptide sequences (TBA-G3FIE-Ac and TBA-G3EIF-Ac) corresponding to a short fragment of the acidic region of the human factor V, which is known to interact directly with exosite I. Surface Plasmon Resonance (SPR) results showed enhanced analytical performances of thrombin with TBA-G3EIF-Ac than with TBA wild-type, reaching a limit of detection as low as 44.9 pM. Electrophoresis mobility shift assay (EMSA) corroborated the SPR results. Molecular dynamics (MD) simulations support experimental evidences and provided further insight into thrombin/TBA-peptide interaction. Our findings demonstrate that the combination of TBA15 with key interacting peptides offers good opportunities to produce sensitive devices for thrombin detection and potential candidates to block thrombin activity. [Display omitted] •Addition of a peptide to the TBA aptamer modulates its affinity for α-thrombin.•The binding of the new TBA-peptide with α-thrombin is characterized by SPR and EMSA.•MD simulations disclose close contacts between the TBA-peptide and α-thrombin.