Engineering nucleic acid structures for programmable molecular circuitry and intracellular biocomputation

Abstract

Nucleic acids have attracted widespread attention due to the simplicity with which they can be designed to form discrete structures and programmed to perform specific functions at the nanoscale. The advantages of DNA/RNA nanotechnology offer numerous opportunities for in-cell and in-vivo applications, and the technology holds great promise to advance the growing field of synthetic biology. Many elegant examples have revealed the potential in integrating nucleic acid nanostructures in cells and in vivo where they can perform important physiological functions. In this Review, we summarize the current abilities of DNA/RNA nanotechnology to realize applications in live cells and then discuss the key problems that must be solved to fully exploit the useful properties of nanostructures. Finally, we provide viewpoints on how to integrate the tools provided by DNA/RNA nanotechnology and related new technologies to construct nucleic acid nanostructure-based molecular circuitry for synthetic biology.

Figure 1 |. Representative examples of promising DNA nanostructures for synthetic biology.

a, 2D DNA crystalline arrays self-assembled from synthetic DNA double-crossover tiles. Scale bar: 300 nm. b, 2D square lattice assembled with 4 × 4 DNA tiles. Scale bar: 100 nm. c, DNA tetrahedral structure. d, Self-assembled 3D DNA crystal from a tensegrity DNA triangle motif. Scale bar: 200μm. e, Hierarchical polyhedral DNA structures. f, 3D structures built with single-stranded tile DNA bricks. g, DNA origami folded by a long single-stranded scaffold DNA and hundreds of short staple DNA oligos. h, Wireframe DNA origami nanostructures with multi-arm junction vertices. Scale bar: 100 nm. i, Hollow DNA box with a controllable lid. j, 3D DNA origami built with multiple pleated layers. Scale bar: 20 nm k, 3D DNA origami with complex curvatures. l, Arbitrary 3D structure built with polyhedral meshes. Scale bar: 50 nm

Figure 2 |. DNA/RNA nanotechnology-enabled toolbox for synthetic circuits.

A diverse set of useful tools have been available; for example, biomolecular scaffolds based on addressable DNA nanostructures, logic units based on DNA strand displacement reactions, DNA nanostructure cell-entry vehicle, HCR-based isothermal construction of DNA structures, targeted editing and error correction based on CRISPR systems, signal readout based on fluorescent RNA motifs and triggers/switches based on siRNAs/microRNAs or riboregulators. ORF, open reading frame.

Figure 3 |. Typical AND gate circuits.

a, A DNAzyme-enabled AND gate. b, A CRISPR-Cas9 system. c, A logic-gated DNA nanorobot that conditionally releases payload molecules^,. d, A combination of riboregulators and recombinases. Based on these principles, other kinds of logic gates (OR, XOR, NOR and so on) and logic-gated cascades can be readily implemented as well.

Figure 4 |. I/O interface scheme for synthetic circuits.

Three main types of components are employed to convert input signals into output signals. Signal transducers like aptamers, riboswitches and ribozymes, detect the inputs (in forms of pH value, UV light, metal ions, small molecules, proteins and so on) and convert them into nucleic acid signal molecules. These DNA/RNA signals undergo information processing using DNA/RNA-based circuitry. Following processing, signals are generated through transcription, translation or DNA lithography and output (in the form of fluorescence, protein products, a coded pattern and so on) from the circuit.

Figure 5 |. Nucleic acid nanostructures as information storage media.

a, Comparison of different information storage media with regard to data density and capacity (Modified from ref. 114). b, Multidimensional information storage can be realized with DNA/RNA nanostructures with various geometrical/topological properties, chemical/biochemical modifications, and dynamic responses.

Figure 6 |. The scheme of an integrated live-cell circuit enabled by DNA/RNA nanotechnology.

Inputs from outside the cell are converted via transducers into DNA/RNA signals that interface directly with intracellular DNA/RNA-based circuitry and memory storage elements. The biosynthetic capabilities of the cell itself are used to fabricate DNA/RNA nanostructures and to produce output signals for a human operator or the intended recipient cells. This live-cell circuit holds promise in applications like diagnosis, therapy, optogenetics, biomanufacturing and robotics.