Single-stranded DNA and RNA origami

Large origami from a single strand Nanostructures created by origami-like folding of nucleic acids are usually formed by base-pairing interactions between multiple strands. Han et al. show that large origami (up to 10,000 nucleotides for DNA and 6000 nucleotides for RNA) can be created in simple shapes, such as a rhombus or a heart. A single strand can be folded smoothly into structurally complex but knot-free structures by using partially complemented double-stranded DNA and the cohesion of parallel crossovers. The use of single strands also enables in vitro synthesis of these structures. Science, this issue p. eaao2648 Large nanostructures of up to 10,000 nucleotides can be formed by folding a single nucleic acid strand. INTRODUCTION Self-folding of an information-carrying polymer into a compact particle with defined structure and function (for example, folding of a polypeptide into a protein) is foundational to biology and offers attractive potential as a synthetic strategy. Over the past three decades, nucleic acids have been used to create a variety of complex nanoscale shapes and devices. In particular, multiple DNA strands have been designed to self-assemble into user-specified structures, with or without the help of a long scaffold strand. In recent years, RNA has also emerged as a unique, programmable material, offering distinct advantages for molecular self-assembly. On the other hand, biological macromolecules, such as proteins (or protein domains), typically fold from a single polymer into a well-defined compact structure. The ability to fold de novo designed nucleic acid nanostructures in a similar manner would enable unimolecular folding instead of multistrand assembly and even replication of such structures. However, a general strategy to construct large [>1000 nucleotides (nt)] single-stranded origami (ssOrigami) remains to be demonstrated where a single-stranded nucleic acid folds into a user-specified shape. RATIONALE The key challenge for constructing a compact single-stranded structure is to achieve structural complexity, programmability, and generality while maintaining the topological simplicity of strand routing (to avoid putative kinetic traps imposed by knots) and hence ensuring smooth folding. The key innovation of our study is to use partially complemented double-stranded DNA or RNA and parallel crossover cohesion to construct such a structurally complex yet knot-free structure that can be folded smoothly from a single strand. RESULTS Here, we demonstrate a framework to design and synthesize a single DNA or RNA strand to efficiently self-fold into an unknotted compact ssOrigami structure that approximates an arbitrary user-prescribed target shape. The generality of the method was validated by the construction of 18 multikilobase DNA and 5 RNA ssOrigami, including a ~10,000-nt DNA structure (37 times larger than the previous largest discrete single-stranded DNA nanostructure) and a ~6000-nt RNA structure (10 times larger than the previous largest RNA structure). The raster-filling nature of ssOrigami permitted the experimental construction of programmable patterns of markers (for example, a “smiley” face) and cargoes on its surface, its single-strandedness enabled the demonstration of facile replication of the strand in vitro and in living cells, and its programmability allowed us to codify the design process and develop a web-based automated design tool. CONCLUSION The work here establishes that unimolecular DNA or RNA folding, similar to multicomponent self-assembly, is a fundamental, general, and practically tractable strategy for constructing user-specified and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology. Folding of DNA or RNA ssOrigami structures. (A) Multiple DNA strands have been designed to self-assemble without (left) or with (middle) a long scaffold strand. Here, we fold single long DNA or RNA strands into target shapes (right). (B) Schematics and atomic force microscopy images of single-stranded DNA (top three rows) and RNA (bottom row) nanostructures. (C) Size comparison between ssOrigami and previously reported single-stranded nucleic acid nanostructures. Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although multicomponent self-assembly has produced complex synthetic nanostructures, unimolecular folding has seen limited progress. We describe a framework to design and synthesize a single DNA or RNA strand to self-fold into a complex yet unknotted structure that approximates an arbitrary user-prescribed shape. We experimentally construct diverse multikilobase single-stranded structures, including a ~10,000-nucleotide (nt) DNA structure and a ~6000-nt RNA structure. We demonstrate facile replication of the strand in vitro and in living cells. The work here thus establishes unimolecular folding as a general strategy for constructing complex and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology.