title: Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.
abstract: Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark.
authors: Daniel M
authors: DM
authors: Dupont
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark.
authors: Cathrine K
authors: CK
authors: Thuesen
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark.
authors: Kenneth A
authors: KA
authors: Bøtkjær
authors: iNANO Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Aarhus, Denmark; Department of Chemistry, Lund University, Lund, Sweden.
authors: Manja A
authors: MA
authors: Behrens
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors: Karen
authors: K
authors: Dam
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark.
authors: Hans P
authors: HP
authors: Sørensen
authors: iNANO Interdisciplinary Nanoscience Center and Department of Chemistry, Aarhus University, Aarhus, Denmark.
authors: Jan S
authors: JS
authors: Pedersen
authors: Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark; Finsen Laboratory, Rigshospitalet and Biotech Research & Innovation Centre, Copenhagen, Denmark.
authors: Michael
authors: M
authors: Ploug
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark.
authors: Jan K
authors: JK
authors: Jensen
authors: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Aarhus, Denmark.
authors: Peter A
authors: PA
authors: Andreasen
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D052156:SELEX Aptamer Technique; D053838:Scattering, Small Angle; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D052157:Aptamers, Nucleotide; D014568:Urokinase-Type Plasminogen Activator