The use of nucleic acids as drugs represents a consistently growing approach. Different therapeutical strategies take advantage
of the biological and biophysical properties of DNA and RNA to properly modulate activity of selected targets. A peculiar characteristic
of these molecules is their structural flexibility which allows them to assume distinct foldings depending upon their sequence and/or environment.
During the last twenty years this has led to the theoretical and experimental development of oligonucleotide aptamers, short sequences
which can recognize a target with specificity and affinity comparable to antibodies. A leading example is represented by the
Thrombin aptamer (15fTBA), a 15-mer DNA selected by its high affinity for the exosite I (fibrinogen binding site) of the coagulation factor.
The very stable protein-DNA complex formation is the result of complementarities between the two macromolecules promoted by
the aptamer sequence and folding as well as of electrostatic interactions generated by the charge balance at the binding site/s. Here, we
investigated the relative role of these contributions and their involvement in defining the biological properties of the resulting complex.
Thus we compared the Thrombin binding and inhibition properties of TBA to those of unrelated single stranded oligonucleotides. Additionally,
the differences between the two protein exosites were assessed by using 29hTBA, a longer (29-mer) aptamer known to bind
exosite II (heparin binding site). A subtle balance of aptamer folding and sequence is shown to cooperate with charge density for effective
and selective recognition of exosite I or exosite II by TBAs.
Keywords: Thrombin, aptamer, single stranded DNA, G-quadruplex, heparin binding site, oligonucleotide, polyanionic, coagulation, thrombomodulin, antibodies
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