title: Distinguishing closely related amyloid precursors using an RNA aptamer.
abstract: Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β2-microglobulin (hβ2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ2m with an EC50 of ∼200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-hβ2m complex, revealing that the aptamer binds to the face of hβ2m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Claire J
authors.initials: CJ
authors.lastname: Sarell
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Theodoros K
authors.initials: TK
authors.lastname: Karamanos
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: White
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: David H J
authors.initials: DHJ
authors.lastname: Bunka
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Arnout P
authors.initials: AP
authors.lastname: Kalverda
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Thompson
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Barker
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. Electronic address: p.g.stockley@leeds.ac.uk.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Stockley
authors.affiliation: Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. Electronic address: s.e.radford@leeds.ac.uk.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D000682:Amyloid; D052157:Aptamers, Nucleotide; D001613:beta 2-Microglobulin
keywords: Amyloid; Amyloid Fibril; Amyloid Precursor; Aptamer; Co-polymerization; Protein Aggregation; Protein Folding; RNA Aptamer; Structural Biology; β2-Microglobulin
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