Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer.

Abstract:

:A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15-0.50 kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by T(m) versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Pasternak A,Hernandez FJ,Rasmussen LM,Vester B,Wengel J

doi

10.1093/nar/gkq823

subject

Has Abstract

pub_date

2011-02-01 00:00:00

pages

1155-64

issue

3

eissn

0305-1048

issn

1362-4962

pii

gkq823

journal_volume

39

pub_type

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