# Bringing proteins into the fold

A molecular origami method uses double-stranded DNA scaffolds and protein staples to create hybrid nanostructures

By Shawn M. Douglas

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA.

Science (2017) 355:1261-2

Molecular engineers have become increasingly adept at repurposing life's building blocks to make custom self-assembled shapes. Because a single drop of solution contains billions of such shapes, DNA origami smiley faces (1), RNA stars (2), and designer protein polyhedra (3) may vastly outnumber most other human-made objects on Earth. These shapes lack immediate practical utility, but they transmit a powerful message: Researchers are beginning to understand how molecules self-assemble. On page 1283 of this issue, Praetorius and Dietz make another leap forward by demonstrating a novel class of nanostructures, namely DNA-protein hybrid shapes (4). This is an important advance because it provides a method to create human-designed shapes out of ingredients that are generally compatible with living systems. In the DNA origami method, a kilobase-long single-stranded DNA (ssDNA) "scaffold" hybridizes with ssDNA oligonucleotide "staples" to form custom assemblies (see the figure). DNA origami provides a user-friendly approach for coaxing biomolecules into specific arrangements with nanoscale precision. It has been used to build imaging probes and prototype drug carriers (5), but early efforts have been limited to in vitro applications. This new work offers a promising route to building custom shapes for in vivo applications.

Praetorius and Dietz have reimagined DNA origami to create a hybrid approach, in which a double-stranded DNA plasmid serves as the scaffold, while engineered transcription activator-like (TAL) effectors act as staples. TAL effectors are proteins that are composed of compact modular DNA nucleotide-binding domains that can be concatenated to bind to arbitrary DNA sequences (6). The authors leveraged TAL effectors to design several custom DNA-binding modules, each with a distinct 20-base-pair DNA substrate. Pairs of TAL effector modules were spliced together to form double-TAL (dT) staple proteins that guided the assembly of the plasmid scaffolds into a variety of custom shapes (see the figure).

TAL effectors are perhaps most notable for their initial promise to supersede zinc-finger nucleases for genome editing (7). However, generating new TAL effectors that bind to a specific DNA sequence is a laborious process, so they were phased into early retirement in favor of clustered regularly interspaced short palindromic repeat (CRISPR)-based proteins that are less cumbersome to work with. Fortunately, Praetorius and Dietz may not have received that memo. In a tour de force, they generated 12 dT staples and used them to fold 16 DNA plasmid scaffolds into different shapes. In doing so, they convincingly show that TAL effectors still hold tremendous potential for biotechnology.

"Their proof of concept offers a tantalizing path toward a new generation of tools that could address important hypotheses about the role of DNA bending and compaction in biology." Indeed, many natural protein complexes that perform gene regulation can be thought of as protein staples that create DNA loops or bends. The authors' approach could be extended to create synthetic enhancers or silencers and eventually a full suite of programmable nanoscale tools for validating models of three-dimensional genome architecture (8).

The hybrid DNA-protein origami method is particularly exciting because it demonstrates that DNA origami is not a one-off method. Instead, Rothemund happened to use DNA to invent a particularly successful instance of a general approach: staple-directed assembly of scaffold templates. It would not be surprising if other permutations of source materials could also be programmed to self-assemble.

## Challenges

Several challenges lie ahead. The method requires a lot of effort to generate new dT staples, which may limit adoption. The DNA-protein hybrid shapes are genetically encoded but have not yet been shown to work in living organisms, unlike alternative RNA-based methods (9) and protein scaffolds (10) that have been used to control metabolic flux in vivo. It may be difficult in general to translate complex self-assembly methods to work in vivo, even though simple DNA nanostructures have been expressed and folded in bacteria (11). On a positive note, the hybrid DNA-protein structures reported by Praetorius and Dietz do not rely on any ssDNA components, which might be a major advantage because custom ssDNA is typically difficult to produce in cells. It will be interesting to see what kinds of functionalization can be added to dT staples, especially because applications in cells may require careful regulation of expression and folding conditions. Fortunately, there will be no shortage of options to explore as molecular engineers get ever closer to biological-level sophistication and complexity.