The thrombin binding aptamer (TBA) is a 15-mer oligonucleotide (ON) which folds into a typical chair-like G-quadruplex structure containing one TGT and two TT loops.(1) It binds thrombin acting as an anticoagulant agent.(2) In our ongoing investigation on the molecular bases of the anti-thrombin activity of TBA, (3,4) we expanded TBA structure-activity relationships (SARs) by synthesizing four new analogues, named TBA-7R(Ph-NO2), TBA-7S(Ph-NO2), TBA-12R(Ph-NO2) and TBA-12S(Ph-NO2), in which, the (R) or (S)-3-(3-nitrophenoxy)propane-1,2-diol moiety replaced, one at a time, the thymine residues at positions 7 and 12. The new derivatives were then tested in vitro using the prothrombin time (PT) coagulation assay. In order to rationalize acquired SARs, a multi‐disciplinary approach including biophysical and computational studies was applied. The evaluation of the anti-thrombin activity showed that all the tested analogues are able to increase the prothrombin time of human plasma at both 2 and 20 μM concentrations. Preliminary results obtained by CD and Electrophoretic Mobility Shift Assay experiments, showed that all the synthesized aptamers are able to fold into G-quadruplex structures in buffered solutions containing Na+ or K+ ions. Analyses of CD melting curves, as well as, preliminary results obtained by computational studies, evidenced that the R or S chirality of (3-nitrophenoxy)propane-1,2-diol at 12 position affected the thermal stability of the resulting TBA-G-quadruplex, while the same effect is not observed at 7 position. Similarly, the relation between G-quadruplex thermal stability and antithrombin activity of the new analogues depends on the position (7 or 12) of the introduced glycerol derivative.

Synthesis, structural behaviors and biological properties of thrombin binding aptamers singly modified with chiral m-nitro-benzene glycidoyl moiety

Maria Scuotto
;
Marco Persico;Concetta Imperatore;Valentina Vellecco;Elena Morelli;Mariarosaria Bucci;Marialuisa Menna;Caterina Fattorusso;Michela Varra
2018

Abstract

The thrombin binding aptamer (TBA) is a 15-mer oligonucleotide (ON) which folds into a typical chair-like G-quadruplex structure containing one TGT and two TT loops.(1) It binds thrombin acting as an anticoagulant agent.(2) In our ongoing investigation on the molecular bases of the anti-thrombin activity of TBA, (3,4) we expanded TBA structure-activity relationships (SARs) by synthesizing four new analogues, named TBA-7R(Ph-NO2), TBA-7S(Ph-NO2), TBA-12R(Ph-NO2) and TBA-12S(Ph-NO2), in which, the (R) or (S)-3-(3-nitrophenoxy)propane-1,2-diol moiety replaced, one at a time, the thymine residues at positions 7 and 12. The new derivatives were then tested in vitro using the prothrombin time (PT) coagulation assay. In order to rationalize acquired SARs, a multi‐disciplinary approach including biophysical and computational studies was applied. The evaluation of the anti-thrombin activity showed that all the tested analogues are able to increase the prothrombin time of human plasma at both 2 and 20 μM concentrations. Preliminary results obtained by CD and Electrophoretic Mobility Shift Assay experiments, showed that all the synthesized aptamers are able to fold into G-quadruplex structures in buffered solutions containing Na+ or K+ ions. Analyses of CD melting curves, as well as, preliminary results obtained by computational studies, evidenced that the R or S chirality of (3-nitrophenoxy)propane-1,2-diol at 12 position affected the thermal stability of the resulting TBA-G-quadruplex, while the same effect is not observed at 7 position. Similarly, the relation between G-quadruplex thermal stability and antithrombin activity of the new analogues depends on the position (7 or 12) of the introduced glycerol derivative.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11588/738137
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