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Distinguishing closely related amyloid precursors using an RNA aptamer

Sarell, CJ, Karamanos, TK, White, SJ, Bunka, DH, Kalverda, AP, Thompson, GS, Barker, AM, Stockley, PG, Radford, SE (2014) Distinguishing closely related amyloid precursors using an RNA aptamer. The Journal of Biological Chemistry, 289 . pp. 26859-26871. ISSN 0021-9258. (doi:10.1074/jbc.M114.595066)

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 ?2-microglobulin (h?2m) can co-polymerize with its N-terminally truncated variant (?N6) 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 h?2m and its amyloidogenic counterpart, ??6, 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 h?2m with an EC50 of ?200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-h?2m complex, revealing that the aptamer binds to the face of h?2m containing the A, B, E, and D ?-strands. In contrast, binding of B6 to ?N6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of h?2m and ?N6 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.

Item Type: Article
DOI/Identification number: 10.1074/jbc.M114.595066
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: G. Thompson
Date Deposited: 23 Jan 2019 21:17 UTC
Last Modified: 30 May 2019 08:47 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/71801 (The current URI for this page, for reference purposes)
Thompson, GS: https://orcid.org/0000-0001-9399-7636
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