Novel Modular DNA Aptamer for Human Thrombin with High Anticoagulant Activity
Alexei Kopylov
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
Abstract: Nucleic acid based aptamers are promising alternative to the antibodies in therapy and diagnostics. Several DNA aptamers against human thrombin have been developed by library selection with SELEX; though no direct structure based drug design for aptamers has been performed yet.
Improved force field for computer molecular dynamics simulations has been developed, and the novel modular DNA aptamer, RA-36, has been created. Comparative studies with two known aptamers, G-quadruplex 15-TBA and 31-TBA, were performed. A thrombin activity assay, as well as specific kinetics calculations, has been originated. Conventional 15-TBA inhibits thrombin at rather high concentrations, 1 - 10 mkM, while RA-36 and 31-TBA are effective at much lower concentrations, 5 - 20 nM. The type of inhibiting is also different which could shed some light on a mode of action of the aptamers on thrombin. Coagulation tests require about 1 mkM RA-36: the aptamer could double APTT; and it shows dose-dependent effect for TT, PT, and APTT. Contrary to 31-ТВА, RA-36 is highly specific for human thrombin in human vs rat tests, therefore a special protocol for aptamer animal testing has been developed. Rat half-life time is about 7 min, which suitable for further application in cardiovascular surgery.
