NMR characterization of the structure and interaction of an RNA aptamer targeting α-synuclein.

α-Synuclein (αSyn) is an intrinsically disordered protein whose aberrant aggregation is a hallmark of Parkinson's disease. C-terminally truncated αSyn variants promote aggregation and fibril formation of full-length αSyn, making them targets for diagnosis and therapy. sh1R6 is an RNA aptamer selected against a C-terminally truncated αSyn variant (αSyn1-95). sh1R6 binds αSyn1-95 with high affinity and suppresses fibril formation of full-length αSyn, but its recognition mechanism remains unclear. In this study, NMR spectroscopy was used to determine the structure of sh1R6 and its mode of interaction with αSyn. The imino-imino NOEs indicated that sh1R6 adopts a stem-loop (hairpin) structure. Upon addition of αSyn1-95, TOCSY spectra of sh1R6 showed pronounced attenuation of H5-H6 correlations of pyrimidine residues in the loop region, thereby identifying the loop and adjacent residues as the primary binding interface. In parallel, addition of sh1R6 to full-length αSyn caused chemical shift perturbations (CSPs) in both the N-terminal and C-terminal regions. These CSPs suggest binding of sh1R6 to the N-terminal region, including the P1 region, a key determinant of αSyn fibril formation, and possible disruption of long-range intramolecular interactions between the N-terminal and C-terminal regions. Thus, in place of the C-terminus, sh1R6 may bind the N-terminal region with higher affinity and mask the N-terminal region including P1 region, thereby preventing αSyn aggregation and fibril formation. Together, these findings define the binding interface between sh1R6 and αSyn1-95 and provide structural and mechanistic insights into how 1R6 modulates αSyn aggregation.