RNA Bind-N-Seq for purified a-Synuclein and BG4 (scFV for G-quadruplex)

Aggregation of a-synuclein may trigger the development of synucleinopathies, a progressive neurodegenerative disease such as Parkinson's disease and Dementia with Lewy bodies. We demonstrate RNA initiates a-synuclein phase transition in vitro, however, RNA binding ability and its motif remain unclear. Here, we indicate purified human a-synuclein protein preferentially binds to guanine-rich RNA sequences by RNA Bind-N-Seq using a pool of random 24-mer RNA oligonucleotides in vitro. Importantly, these hit motifs were required consecutive guanines not random, suggesting that RNA G-quadruplexes binds to a-synuclein. We also confirmed that GC content and skew of a-synuclein-enriched RNA sequences were similar to that of BG4 (G-quadruplex antibody)-enriched RNA sequences. Taken together, these data suggest that a-synuclein binds to RNA-formed G-quadruplex. Overall design: To investigate a-synuclein-bound RNA motifs, human a-synuclein was incubated with 24-mers randomized RNA pools. BG4 known as G-quadruplex antibody is utilized for positive control.