Alpha-Synuclein Modulates the Physical Properties of DNA

Abstract

Fundamental research on Parkinson's disease (PD) most often focuses on the ability of α-synuclein (aS) to form oligomers and amyloids, and how such species promote brain cell death. However, there are indications that aS also plays a gene-regulatory role in the cell nucleus. Here, the interaction between monomeric aS and DNA in vitro has been investigated with single-molecule techniques. Using a nanofluidic channel system, it was discovered that aS binds to DNA and by studying the DNA-protein complexes at different confinements we determined that aS binding increases the persistence length of DNA from 70 to 90 nm at high coverage. By atomic force microscopy it was revealed that at low protein-to-DNA ratio, the aS binding occurs as small protein clusters scattered along the DNA; at high protein-to-DNA ratio, the DNA is fully covered by protein. As DNA-aS interactions may play roles in PD, it is of importance to characterize biophysical properties of such complexes in detail.

Keywords: DNA; biochemistry; nanofluidics; proteins; synuclein.

Figure 1

(A) Schematic illustration of the nanofluidic chip design (left). The channel system comprises pairs of micro‐channels, spanned by an array of straight nanochannels. The cartoon shows DNA polymers confined inside a nanochannel. DNA will be partially stretched along the nanochannel, with an extension R_∥, shorter than its contour length L. (B) Montage of fluorescence images of λ‐DNA with aS. From up to bottom: 0, 1, 5, 15 and 40 μm aS. The YOYO:bp ratio is 1:50. (C) Extension distribution of λ‐DNA molecules in 150×100 nm² nanochannels at different concentrations of aS.

Figure 2

(A) Relative extension R_∥ L⁻¹ of λ‐DNA in 1×TE buffer in 150×700 nm² channels (•, red) and 150×100 nm² channels (▪, black), and λ‐DNA in 1×TE buffer with 30 mm NaCl in 150×100 nm² channels (Δ, blue), versus the α‐synuclein concentration. (B) The ratio between the extension of λ‐DNA molecules in 150×100 nm² and 150×700 nm² channels versus aS concentration. (C) Fitted data for DNA persistence length versus aS concentration. The dashed and solid curves are drawn as an aid to the eye.

Figure 3

Tapping mode atomic force microscopy images of DNA (1 kb, 5 μm base pairs) with aS at a concentration of (A) 0 μm, (B) 0.1 μm and (C) 40 μm. The scale bar is 100 nm. The line in each panel corresponds to the height profile of the molecule shown to the right. Mica (A) and silica (B and C) surfaces were used (see Materials and Methods for details).