Premium
Selection of DNA Aptamer That Blocks the Fibrillogenesis of a Proteolytic Amyloidogenic Fragment of β 2 m
Author(s) -
Fukasawa Ka,
Higashimoto Yuichiro,
Ando Yukio,
Motomiya Yoshihiro
Publication year - 2018
Publication title -
therapeutic apheresis and dialysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.415
H-Index - 53
eISSN - 1744-9987
pISSN - 1744-9979
DOI - 10.1111/1744-9987.12591
Subject(s) - aptamer , thioflavin , fibrillogenesis , clone (java method) , amyloid (mycology) , amyloidosis , in vitro , congo red , biophysics , microbiology and biotechnology , chemistry , biochemistry , dna , medicine , biology , pathology , disease , organic chemistry , adsorption , alzheimer's disease
Dialysis‐related amyloidosis (DRA) is a severe complication of hemodialysis that results in progressive destruction of bones and joints. Elevated concentrations of the β 2 ‐microglobulin (β 2 m) level in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in osteoarticular tissues. β 2 m lacking the N‐terminal six residues of the mature protein (ΔN6β 2 m) constitutes 25–30% of β 2 m in ex vivo DRA amyloid. Unlike full‐length wild‐type β 2 m, ΔN6β 2 m forms amyloid fibrils at neutral pH in vitro. However, the role of ΔN6β 2 m in DRA is, at present, poorly understood. In the present study, we screened novel phosphorothioate‐modified aptamers directed against ΔN6β 2 m using combinatorial chemistry in vitro. We identified 11 ΔN6β 2 m aptamers; among the identified aptamers, clone #2, #8, and #10 aptamers had higher binding affinity to ΔN6β 2 m than the others. Biolayer interferometry analysis revealed that K D values of clone #2, #8, and #10 aptamers were 56, 23, and 44 nM, respectively. Furthermore, the clone #8 aptamer inhibited fibril formation in a dose‐dependent manner, as assessed by Thioflavin T fluorescence assay. Fibrils formed from ΔN6β 2 m bind to Congo red, displaying changes in the absorbance spectrum of the dye characteristic of binding to amyloid fibrils, which was completely blocked by treatment with clone #8 aptamer. These results suggest the potential of ΔN6β 2 m aptamers as tools for elucidating co‐assembly mechanisms in amyloid formation.