Novel Modular DNA Aptamer for Human Thrombin with High Anticoagulant Activity

E.      Zavyalova; A.      Golovin; R.      Reshetnikov; N.      Mudrik; D.      Panteleyev; G.      Pavlova; A.      Kopylov

Abstract

Aptamers based on nucleic acids are a promising alternative to antibodies in therapy and diagnostics. Several DNA aptamers against human thrombin have been developed by selection from random libraries: a 15-mer and its derivatives, a 29-mer, and a 31-mer. Some of them are patented and already under clinical trial. The 15-mer structure was determined by X-ray and NMR and turned out to be a monomolecular antiparallel G-quadruplex. The other aptamers mentioned above have higher inhibitory activity than the initial 15-mer, but there are not yet structural data explaining this phenomenon. Here, the initial 15-mer, 31-mer, and novel RA-36 aptamers are compared to establish the structure – function correlation, providing a solid ground for further rational aptameric drug design. For the molecular dynamic simulation of aptamers, the force field parmbsc0 was ported onto GROMACS, and the main stabilizing parameters were revealed, leading to the novel DNA aptamer RA-36. The functional properties of the DNA aptamers were studied by conventional coagulation tests, which do not directly elucidate the mechanism of thrombin inhibition by aptamers. Improved turbidimetric measurements provided data to develop detailed kinetics showing that the 31-mer and RA-36, in contrast to the 15-mer, are competitive inhibitors. These data revealed RA-36 to be an efficient thrombin inhibitor with a dose-dependent effect. Animal tests of the studied DNA aptamers suggested an unexpected species-specificity of the novel RA-36.

Keywords: Anticoagulant, coagulation, hemostasis, G-quadruplex, molecular dynamic simulation, pharmacodynamics, pharmacokinetics, thrombin, thrombin binding DNA aptamer, thrombin inhibitor, turbidimetry, anti-inflammatory properties