title: Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer.
abstract: Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.
authors.affiliation: Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Troels B
authors.initials: TB
authors.lastname: Jensen
authors.forename: Jonas R
authors.initials: JR
authors.lastname: Henriksen
authors.forename: Bjarne E
authors.initials: BE
authors.lastname: Rasmussen
authors.forename: Lars M
authors.initials: LM
authors.lastname: Rasmussen
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Andresen
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: Anna
authors.initials: A
authors.lastname: Pasternak
mesh_terms: D000925:Anticoagulants / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001777:Blood Coagulation; D002942:Circular Dichroism; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D054856:G-Quadruplexes; D006358:Hot Temperature; D058990:Molecular Targeted Therapy; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry* / Q000378:metabolism; D009841:Oligonucleotides / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D010879:Piperazines / Q000737:chemistry; D013053:Spectrophotometry; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism; D013918:Thrombin Time; D014498:Uracil / Q000737:chemistry
chemical_list: D000925:Anticoagulants; D052157:Aptamers, Nucleotide; D009696:Nucleic Acids; D009841:Oligonucleotides; D010879:Piperazines; C081676:thrombin aptamer; D014498:Uracil; D013917:Thrombin