Published: January 10, 2011 r2011 American Chemical Society 657 dx.doi.org/10.1021/nl1037769 | Nano Lett. 2011, 11, 657–660 LETTER pubs.acs.org/NanoLett DNA-Templated Protein Arrays for Single-Molecule Imaging Daniele N. Selmi, † Roslin J. Adamson, ‡ Helen Attrill, ‡ Alan D. Goddard, ‡ Robert J. C. Gilbert,* ,§ Anthony Watts,* ,‡ and Andrew J. Turberfield* ,† † Department of Physics, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, U.K. ‡ Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. § Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, U.K. b S Supporting Information ABSTRACT: Single-particle electron cryomicroscopy permits structural characterization of noncrystalline protein samples, but throughput is limited by problems associated with sample preparation and image processing. Three-dimensional density maps are reconstructed from high resolution but noisy images of individual molecules. We show that self- assembled DNA nanoaffinity templates can create dense, nonoverlapping arrays of protein molecules, greatly facilitating data collection. We demonstrate this technique using a G-protein-coupled membrane recep- tor, a soluble G-protein, and a signaling complex of both molecules. KEYWORDS: Single-particle electron cryomicrosopy, DNA templates, nanostructure, nanoaffinity, G-protein-coupled membrane receptor S ingle-particle electron cryomicroscopy (cryo-EM) allows direct visualization of biomolecules, flash-frozen in a solution suspended across holes in a carbon film. 1 This technique is particularly promising for hard-to-crystallize membrane proteins and protein complexes. 2 However, high beam currents damage samples and high protein densities lead to aggregation: through- put and resolution are severely limited by the need to identify, orient, and average 10 4 -10 6 noisy, low-dose projection images of sparse, randomly distributed particles. 3 By attaching the target protein to a self-assembled 2D DNA template, we are able to create arrays of protein molecules that greatly simplify data acquisition. The proposal that a synthetic protein crystal assembled on a 3D DNA template could be used for X-ray diffraction 4 is a driving force in the development of DNA nanotechnology. While this goal has yet to be realized, DNA-based approaches have been successfully employed to investigate protein structures: liquid- crystalline DNA nanotubes have been used to orient membrane proteins for structure determination by NMR, 5 and we have used a DNA-templated 2D crystal to obtain a low-resolution projec- tion map of the soluble protein RuvA by cryo-EM. 6 Here we describe a fundamentally different use of a self-assembled DNA structure: we attach the target protein to a 2D DNA template, thereby creating dense, nonoverlapping arrays of protein mole- cules suitable for imaging by cryo-EM. Use of flexible linkers ensures that images corresponding to a wide range of molecular orientations are obtained. We demonstrate the technique using samples that span the range of potential targets for single-particle cryo-EM: a soluble, asymmetric guanine nucleotide-binding protein (GR i1 ); a neuropeptide-binding G-protein-coupled membrane receptor (GPCR), the rat neurotensin receptor type 1 (NTS1); 7 and a complex of the two. The template 6 consists of three sets of parallel DNA helices woven, as in kagome basketwork, to produce a trigonal 2D crystal (Figure 1). The four component oligonucleotides were designed to form a Holliday junction with four double-helical arms, each with a 6-nucleotide single-stranded sticky end. Hybridization of complementary sticky ends assembles these motifs into the crystal. Figure 1d and Figure 2a show the template: an unbroken single layer extends across several holes of a holey carbon EM grid (the measured lattice constant is 14 nm). One oligonucleo- tide is modified such that the DNA template presents triangular clusters of binding sites for the protein arranged on a hexagonal lattice. The guanine nucleotide binding protein GR i1 is a small (40 kDa), asymmetric, soluble, protein whose structure in its GDP- bound form has been determined to 2.4 Å (PDB: 1AS3). GDP- bound GR i1 was bound to the DNA template through a Ni 2þ - mediated interaction between a N-terminal (His) 6 affinity tag and tris-nitrilotriacetic acid (tris-NTA) conjugated to a template oligonucleotide. 8 Tris-NTA-functionalized templates were incu- bated with protein, pipetted onto a holey carbon grid, washed briefly by inversion on a 20 μL droplet of distilled water, flash- frozen in liquid ethane, and imaged using a field-emission gun Received: October 27, 2010 Revised: December 24, 2010