Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Nucleic acid aptamers are effective regulatory agents for molecular intervention.
• Serine proteases, like urokinase-type plasminogen activator (uPA), are crucial in physiological and pathophysiological processes.
• Understanding aptamer-protease interactions is key for developing targeted therapies.

Purpose of the Study:

• To delineate the binding sites of two therapeutic aptamers, upanap-12 and upanap-126, on the serine protease uPA.
• To investigate the impact of aptamer binding on uPA's molecular interactions.
• To model the aptamer-protein complex and understand inhibition mechanisms.

Main Methods:

• Site-directed mutagenesis to map aptamer binding sites on uPA.
• Small-angle X-ray scattering (SAXS) analysis.
• Characterization of aptamer binding impact on uPA interactions with plasmin, PAI-1, uPAR, and LRP-1A.

Main Results:

• Upanap-126 binds near the C-terminal α-helix of pro-uPA.
• Upanap-12 binds to the β-hairpin of the growth factor domain and the kringle domain of uPA.
• A model of the upanap-12:pro-uPA complex was constructed, revealing extensive steric interference.

Conclusions:

• Aptamer binding sites on uPA were identified, elucidating specific interaction points.
• The study highlights how aptamer size, shape, and uPA's domain structure contribute to inhibition via steric hindrance.
• Findings advance the understanding of aptamer-based inhibition strategies for serine proteases.