Selection of single-stranded DNA molecules that bind and inhibit human thrombin

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Aptamers are nucleic acid molecules with high specificity for molecular targets.
• Previous aptamer development primarily utilized double-stranded DNA or single-stranded RNA.
• Single-stranded DNA aptamers and targets not interacting physiologically with nucleic acids remained largely unexplored.

Purpose of the Study:

• To investigate the potential of single-stranded DNA (ssDNA) as aptamers.
• To isolate and characterize ssDNA aptamers targeting thrombin, a critical protease in the coagulation cascade.
• To evaluate the functional inhibitory capacity of these aptamers on thrombin activity.

Main Methods:

• In vitro selection and polymerase chain reaction (PCR) were employed to enrich aptamer candidates from a random DNA pool.
• Sequencing of isolated aptamers identified conserved regions crucial for target binding.
• Binding affinities were quantified using nanomolar (nM) concentration ranges.
• In vitro assays assessed the inhibition of thrombin-catalyzed fibrin clot formation.

Main Results:

• Successfully isolated single-stranded DNA aptamers specific for the target protein thrombin.
• Achieved binding affinities for thrombin aptamers in the range of 25-200 nM.
• Identified a conserved 14-17 base region in 32 sequenced thrombin aptamers.
• Demonstrated that several ssDNA aptamers inhibited thrombin's fibrin-clotting activity at nanomolar concentrations in purified and plasma samples.

Conclusions:

• Single-stranded DNA can function as effective aptamers against protein targets.
• The identified ssDNA aptamers exhibit high affinity and functional inhibitory activity against thrombin.
• These findings expand the scope of aptamer technology for targeting proteins in biological systems, particularly in coagulation research.