Computing in mammalian cells with nucleic acid strand exchange

Abstract

DNA strand displacement has been widely used for the design of molecular circuits, motors, and sensors in cell-free settings. Recently, it has been shown that this technology can also operate in biological environments, but capabilities remain limited. Here, we look to adapt strand displacement and exchange reactions to mammalian cells and report DNA circuitry that can directly interact with a native mRNA. We began by optimizing the cellular performance of fluorescent reporters based on four-way strand exchange reactions and identified robust design principles by systematically varying the molecular structure, chemistry and delivery method. Next, we developed and tested AND and OR logic gates based on four-way strand exchange, demonstrating the feasibility of multi-input logic. Finally, we established that functional siRNA could be activated through strand exchange, and used native mRNA as programmable scaffolds for co-localizing gates and visualizing their operation with subcellular resolution.

Complexity from Simple Building Blocks: Engineering Large-scale Information-processing Networks from Molecules

“…To give an example, it took more than a decade of intense effort in a number of research labs to lead to the first report on DNA-based logic gates operating in mammalian cells a few months ago. [13] 3. Molecular Computing as an Enabling Te chnology for Drug Discovery…”

Precisely Tailored DNA Nanostructures and their Theranostic Applications

“…(f) DNA origami‐based nanorobot for logic gated drug release (reproduced from Ref). (g) RNA interference based on strand exchange‐mediated logic calculations in living cells (reproduced from Ref). (h) Oligonucleotide nano‐vehicle with dual locks for logic gated delivery of siRNAs (reproduced from Ref ).…”

“…They delivered DNA nanostructure‐based logic units into mammalian cells to realize AND and OR logic gates responsive to native mRNAs. The positive output of them led to the activation of a siRNA, which effectively knock down the GFP expression in target cells (Figure g) …”

Mammalian synthetic biology in the age of genome editing and personalized medicine

“…One example makes use of conditionally active “splice-switching” oligos that either hybridizes with pre-mRNA or becomes cleaved in the presence of light, thereby altering gene expression by masking or revealing splice sites, respectively [30]. Additional examples of RNA-based regulatory schemes include the use of four-way nucleic acid strand exchange to report intracellular mRNA levels [31], and the programming of ribozyme switches to exert cell-cycle control in response to the chemical ligand theophylline [32]. Furthermore, coupling a β-catenin–binding RNA aptamer to microRNA (miRNA) targeted against a green fluorescent protein (GFP) mRNA construct has been shown to enable quantification of nuclear β-catenin concentrations [33].…”