Programmable Nanodisc Patterning by DNA Origami

Abstract

Nanodiscs (ND) are soluble phospholipid bilayers bounded by membrane scaffold proteins; they have become invaluable in the study of membrane proteins. However, this multifunctional tool has been used individually, and applications involving multiple NDs and their interactions have fallen far behind their counterpart membrane model system: liposomes. One major obstacle is the lack of reliable methods to manage the spatial arrangement of NDs. Here we sought to extend the utility of NDs by organizing them on DNA origami. NDs constructed with DNA-anchor amphiphiles were placed precisely and specifically into these DNA nanostructures via hybridization. Four different tethering strategies were explored and validated. A variety of geometric patterns of NDs were successfully programmed on origami, as evidenced by electron microscopy. The ND ensembles generated in this study provide new and powerful platforms to study protein-lipid or protein-protein interactions with spatial control of membranes.

Keywords: DNA origami; DNA−protein conjugation; membrane proteins; nanodisc.

Figure 1.

Construction of DNA-tethered nanodiscs (DND). (a) DNA conjugation reaction with cholesterol (1), phospholipid (2), protein transmembrane domain (3), or MSP (4). (b) SDS-PAGE stained for DNA (left) or protein (right) of each product, (1)-(4). (c) DND formation (DND1-DND4) by incorporating corresponding DNA-anchor amphiphile ((1)-(4)) in the ND reconstitution mixture. (d) SDS-PAGE and TEM characterization of DND1-DND4. Homogeneous circular particles in TEM images (right) indicate proper ND formation. Protein ladder bands (from top to bottom): 250, 130, 100, 70 (red), 55, 35, 25 (red), 15 kDa. Scale bar: 25 nm.

Figure 2.

DND patterning on DNA origami. Schematics and representative TEM images are shown for each case. (a) Attachment yield analysis of single DNDs on origami. Successful attachment was observed for >75% of V-shaped origami structures for all four types of DNDs (inset), validating the strategy of tethering NDs to DNA for spatial arrangement. (b) A circle of NDs was created by a DNA nanocage with handles on one ring. Again, all four types of DNDs (DND1–4) were successfully organized. (c) More patterning of NDs by a nanocage: semicircle (left), square (middle), and two parallel rings (right). Scale bars: 50 nm.

Figure 3.

Versatility of origami-templated ND patterning. Schematics (left) and representative TEM images (right) are shown for each case. (a) Linear ND array arranged by an origami rod with handles on one side. (b) DNA-encoded ND placed on specific arm of a strutted V-origami. (c) Hierarchical stacking of a circle of NDs on nanocage. Scale bars: 50 nm.

Scheme 1.

ND patterning by DNA origami. DNA-anchor amphiphiles were generated and added into reconstitution mixtures to form DNDs, which were templated by handle-equipped origami structures via DNA hybridization.