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

A. Aviñó, A. F. Jorge, C. S. Huertas, T. F.G.G. Cova, A. Pais, L. M. Lechuga, R. Eritja, C. Fabrega

Biochimica et Biophysica Acta (BBA) - General Subjects, 1863, 2019, 1619-1630 - DOI: 10.1016/j.bbagen.2019.06.014

http://doi.org/10.1016/j.bbagen.2019.06.014

Abstract: 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

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