Distinguishing closely related amyloid precursors using an RNA aptamer

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.