Production and Characterization of RNA Aptamers Specific for Amyloid Fibril Epitopes

Abstract

One of the most fascinating features of amyloid fibrils is their generic cross-␤ architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloidlike fibrils formed in vitro from ␤ 2 -microglobulin (␤ 2 m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K D ϳ nM) to ␤ 2 m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of ␤ 2 m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.A number of proteins and peptides undergo specific aggregation in vivo, leading to a range of pathological disorders, collectively known as amyloidoses (1, 2). These diseases are characterized by the deposition of normally soluble proteins or peptides into insoluble fibrillar arrays with a cross-␤ architecture (1, 2). About 30 different proteins have been identified as the fibrillar component of human amyloid deposits to date, although studies have shown that amyloid-like fibrils can be generated in vitro from virtually any protein sequence under suitable experimental conditions (3, 4). Remarkably, although amyloid and amyloid-like fibrils are formed from diverse precursor proteins with unrelated primary sequences and tertiary folds, they all adopt a cross-␤ molecular architecture, identified by x-ray fiber diffraction (5, 6) and bind a number of histological dyes, such as thioflavin T (ThT) and Congo Red (7,8), the latter showing a characteristic optical anisotropy with apple-green birefringence when viewed using cross-polarized light (9). The ubiquitous ability of amyloid fibrils to bind serum amyloid P component (10, 11), glycosaminoglycans and apolipoprotein E (12), together with the finding that antibodies (e.g. WO1) raised against A␤-(1-40) amyloid fibrils are also able to recognize amyloid fibrils formed from a wide variety of proteins and peptides, unrelated in sequence and structure (13), reinforces the view that amyloid fibrils possess a common core structure. Despite the immense interest in this field (1, 14), the mechanism of how normally soluble proteins or peptides are transformed into the ordered cross-␤ structure of amyloid is largely unknown, although partial denaturation of the native protein, or folding of disordered states to...

Aptamers

“…(8) HFIP-treated lysozyme and IAPP contained sufficient β-sheet content as inferred from their recognition by the tested aptamers and the naïve library. Our observation of non-specific reactivity with fibrils of selected and tested aptamers, which is reminiscent of similar findings in previous studies [62,125,134,136], suggest that aptamers (and naïve libraries of oligonucleotides) likely recognize potentially common aptatopes [62,153,160].…”

“…However, in PD, the presynaptic neuronal, 140 amino acid-long protein α-synuclein and not Aβ has been identified as a possibly responsible agent for the pathogenesis [136]. The aggregation of α-synuclein proteins into oligomers eventually leads to the formation of fibrils which then accumulate in cytosolic filamentous inclusions called Lewy bodies, which are the hallmark of PD [137].…”

“…An RNA aptamer selected for the recombinant bovine PrP reportedly recognized bovine PrP-β [134]-a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils [135]. Bunka et al generated aptamers for monomeric and several forms of fibrillar β2-microglobulin [136]. These aptamers were found to bind also fibrils of other amyloidogenic proteins, including apomyoglobin, Aβ40, transthyretin, or lysozyme, in addition to those of β2-microglobulin [136].…”

Hemodialysis‐Related Amyloidosis

“…However, whilst the antioligomer antibody also binds WL fibrils, only weak binding is found in LS fibrils, suggesting that the epitope recognized by this antibody may be masked in the higher-order assembly. Small structured RNA molecules known as aptamers have also been produced and have been demonstrated to bind to both the acid unfolded state and WL and LS fibrils of b 2 m with nanomolar affinity [81]. Specific aptamers directed against the WL and LS fibrils do not bind the acid unfolded state and hence recognize fibril-specific epitopes [81].…”

“…Small structured RNA molecules known as aptamers have also been produced and have been demonstrated to bind to both the acid unfolded state and WL and LS fibrils of b 2 m with nanomolar affinity [81]. Specific aptamers directed against the WL and LS fibrils do not bind the acid unfolded state and hence recognize fibril-specific epitopes [81]. The distinct kinetics and avidity of the different fibril forms for the different aptamers implies either that the anti-WL and anti-LS aptamers bind distinct epitopes, or that the solvent exposure of a common epitope differs substantially in the different fibril types.…”