Research Article

Self-assembly of genetically encoded DNA-protein hybrid nanoscale shapes

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Science  24 Mar 2017:
Vol. 355, Issue 6331, eaam5488
DOI: 10.1126/science.aam5488

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Protein-folded DNA nanostructures

A wide variety of DNA nanostructures have been assembled by folding long DNA single strands with short DNA staples. However, such structures typically need annealing at elevated temperatures in order to form. To accommodate the formation of such structures in living cells, Praetorius and Dietz developed an approach in which custom protein staples based on transcription activator–like effector proteins fold double-stranded DNA templates (see the Perspective by Douglas). The structures folded into user-defined geometric shapes on the scale of tens to hundreds of nanometers. These nanostructures could self-assemble at room temperature in physiological buffers.

Science, this issue p. eaam5488; see also p. 1261

Structured Abstract

INTRODUCTION

Controlling the spatial arrangement of functional components in biological systems on the scale of higher-order macromolecular assemblies is an important goal in synthetic biology. Achieving this goal could yield new research tools and pave the way for interesting applications in health and biotechnology. DNA origami enables constructing arbitrary shapes on the desired scale by folding a single-stranded DNA “scaffold” into user-defined shapes using a set of “staple” oligonucleotides, but single-stranded DNA is typically not available in living cells, and the structures usually assemble only at nonphysiological temperatures. The scope of functions that can be fulfilled by DNA itself appears so far still limited. Proteins, on the other hand, offer a large variety of functionalities and are easily accessible in cells through genetic encoding, but designing larger structural frameworks from proteins alone remains challenging.

RATIONALE

Here, we reimagined DNA origami to explore the possibility for using a set of designed proteins to fold a double-stranded DNA template into user-defined DNA-protein hybrid objects with dimensions on the desired 10- to 100-nm scale. To realize our idea, we require synthetic DNA “looping” proteins that can link two user-defined double-helical DNA sequences, and we need to determine suitable rules for arranging both the template and multiple of such staple proteins in the context of a larger target structure. Transcription activator–like (TAL) effector proteins are produced by plant pathogenic bacteria and injected into host cells, where they bind to specific promoter regions, thus controlling the expression of target genes. The DNA recognizing part of a TAL effector consists of an array of repeat subunits that binds to the major groove of double-helical DNA, thus forming a superhelix. Each repeat subunit comprises ~34 amino acids and recognizes a single DNA base pair. Because of their modular architecture, TAL effectors can be engineered to bind to user-defined DNA sequences, and this technique is currently being exploited for genome engineering applications. For constructing the staple proteins, we therefore chose the DNA-recognizing domains of TAL effectors.

RESULTS

We characterized the DNA-recognizing domains of the TAL effectors with respect to binding affinity and sequence specificity. To construct the staple proteins, we fused two TAL proteins via a custom peptide linker and tested for the ability to connect two separate double-helical DNA domains. For creating larger objects containing multiple staple protein connections, we identified a set of rules regarding the optimal spacing between these connections. On the basis of these rules, we could create megadalton-scale objects that realize a variety of structural motifs, such as custom curvatures, vertices, and corners. Each of those objects was built from a set of 12 double-TAL staple proteins and a template DNA double strand with designed sequence. We also tested design principles for multilayer structures with enhanced rigidity. All components of our nanostructures can be genetically encoded and self-assemble isothermally at room temperature in near-physiological buffer conditions. The staple proteins used in this work also carried a green fluorescent protein domain that serves as a placeholder for a variety of functional protein domains that can be genetically fused to the staple proteins. We were also able to demonstrate formation of our structures starting from genetic expression in a one-pot reaction mixture that contained the double-stranded DNA scaffold, the genes encoding the staple proteins, RNA polymerase, ribosomes, and cofactors for transcription and translation. Successful self-assembly of our hybrid nanostructures was confirmed using transmission electron microscopy.

CONCLUSION

By using our system of designing double-TAL staple proteins that fold a template DNA double strand, researchers can control the spatial arrangement of protein domains in custom geometries. Our system should have a good chance to work inside cells, given the success of our in vitro expression experiments and considering that the DNA binding properties of TAL are preserved inside cells, as seen in gene editing experiments with TAL-based endonucleases. Because TAL-based staple proteins can be tailored to specifically recognize any desired DNA target sequence, these proteins could then be used to create custom structures and loops in genomic DNA to study the relation between genome architecture and gene expression, or to position proteins involved in other intracellular processes in user-defined ways.

Schematic illustration of the self-assembly of DNA-protein hybrid objects from a set of 12 double-TAL staple proteins and a template DNA double strand.

Depending on the sequence of the template, the resulting object may be a square (top) or a double-ring circle (bottom). In total, we designed 18 different protein hybrid objects using this principle. Scale bar, 20 nm.

Abstract

We describe an approach to bottom-up fabrication that allows integration of the functional diversity of proteins into designed three-dimensional structural frameworks. A set of custom staple proteins based on transcription activator–like effector proteins folds a double-stranded DNA template into a user-defined shape. Each staple protein is designed to recognize and closely link two distinct double-helical DNA sequences at separate positions on the template. We present design rules for constructing megadalton-scale DNA-protein hybrid shapes; introduce various structural motifs, such as custom curvature, corners, and vertices; and describe principles for creating multilayer DNA-protein objects with enhanced rigidity. We demonstrate self-assembly of our hybrid nanostructures in one-pot mixtures that include the genetic information for the designed proteins, the template DNA, RNA polymerase, ribosomes, and cofactors for transcription and translation.

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