Single molecule DNA origami nanoarrays with controlled protein orientation

Abstract

The nanoscale organization of functional (bio)molecules on solid substrates with nanoscale spatial resolution and single-molecule control—in both position and orientation—is of great interest for the development of next-generation (bio)molecular devices and assays. Herein, we report the fabrication of nanoarrays of individual proteins (and dyes) via the selective organization of DNA origami on nanopatterned surfaces and with controlled protein orientation. Nanoapertures in metal-coated glass substrates were patterned using focused ion beam lithography; 88% of the nanoapertures allowed immobilization of functionalized DNA origami structures. Photobleaching experiments of dye-functionalized DNA nanostructures indicated that 85% of the nanoapertures contain a single origami unit, with only 3% exhibiting double occupancy. Using a reprogrammed genetic code to engineer into a protein new chemistry to allow residue-specific linkage to an addressable ssDNA unit, we assembled orientation-controlled proteins functionalized to DNA origami structures; these were then organized in the arrays and exhibited single molecule traces. This strategy is of general applicability for the investigation of biomolecular events with single-molecule resolution in defined nanoarrays configurations and with orientational control of the (bio)molecule of interest.

Minimizing Structural Heterogeneity in DNA Self-Assembled Dye Templating via DNA Origami-Tuned Conformations

“…The successful formation of HJ-out with the designed conformations was confirmed through AFM characterization in liquid (Figures B and S4–S10) and quantified by a high attachment yield of the HJ on the surface of DOC for the three conformations (Tables and S1–S3). The yield of at least 69% ( HJ-120° ) is comparable to the attachment yield of proteins organized on the surface of DOCs. − Although up to three HJs may bind simultaneously to the DOC, AFM imaging consistently showed only one HJ attached to the surface of the DOC. This consistent result can be ascribed to the idea that, given sufficient incubation time, the free arms of a bound HJ would replace the anchored arms of other bound HJs.…”

“…While the second strategy ( HJ-in ) was achieved via a single-pot reaction, our first strategy ( HJ-out ) required the separated assembly of the HJ, which could be leveraged to study the folding pathways of DNA in HJs and crossovers by changing the preparation procedure. Furthermore, the second strategy can be useful to achieve selected conformations and orientations of biomolecules, such as proteins presenting designed orientation-controlled reaction sites for DNA handles, , involved in photo/chemical applications including light-harvesting and catalysis.…”

Programmed Self-Assembly of DNA Nanosheets with Discrete Single-Molecule Thickness and Interfacial Mechanics: Design, Simulation, and Characterization

“…Total internal reflection fluorescence (TIRF) microscopy, a technique that allows characterizing fluorescence emitted within a ~100 nm evanescence region standing from a thin glass coverslip surface [8,13,14,28], was used to monitor the presence of F-units and potential associated superstructures at the oil/aqueous interface (Figure 5f). The results of TIRF microscopy confirmed that AB duplexes were anchored to C chol (at the oil/aqueous interface), but it did not inform us whether a superstructure with a single-molecule DNA thickness was present, as instead observed in Sections 3.3 and 3.4.…”

“…Small DNA strands protruding from each unit, called sticky ends, allow the selective binding of units via DNA base pairing. Whereas using large and relatively robust DNA units, such as DNA origami, provide abundant space per unit for molecular arrangement and functionality [8,9,[12][13][14][15][16]21,22], small units provide simplicity and a large quantity of units per volume as they are composed of a few addressable DNA strands [3,20,[23][24][25][26][27]. The addressability of such strands may provide a high density of functional moieties per unit in a large nanosheet, which can help with promoting adhesion to surfaces.…”