Engineering synthetic RNA devices for cell control

Abstract

The versatility of RNA in sensing and interacting with small molecules, proteins and other nucleic acids while encoding genetic instructions for protein translation makes it a powerful substrate for engineering biological systems. RNA devices integrate cellular information sensing, processing and actuation of specific signals into defined functions and have yielded programmable biological systems and novel therapeutics of increasing sophistication. However, challenges centred on expanding the range of analytes that can be sensed and adding new mechanisms of action have hindered the full realization of the field's promise. Here, we describe recent advances that address these limitations and point to a significant maturation of synthetic RNA-based devices.

Fig. 1 |. RNA cellular functions.

a | Following transcription, mRNAs are spliced by the spliceosome, a complex of small nuclear RNAs (snRNA) and proteins, and matured by the addition of markers such as a 5′ cap (green circle) and polyA tail (green line)^,. Outside the nucleus, the canonical function of RNA is the combination of tRNAs, ribosomal RNAs (rRNAs) (with accompanying ribosomal proteins, making up the ribosome) and mRNAs to form translational complexes that produce most cellular proteins. Circular RNAs (circRNAs) are RNA molecules that form naturally via lariat intermediates as a splicing by-product. RNA regulation of cellular processes can be accomplished by circRNAs, as well as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) that can regulate levels of exogenous RNA, endogenous RNA and protein expression^,. b | RNA device engineering. Parts for RNA devices can be obtained by mining natural sequences, selection experiments or rational design of sequences. Sensing and actuating components can be characterized and combined to form an RNA device with both sensing and actuation capabilities.

Fig. 2 |. RNA devices enable diverse applications.

Versatility of RNA highlighted in the wide application space of RNA devices ranging from addressing bottlenecks in basic research (part A), to providing advancements in biomanufacturing (part B) to propelling new frontiers in human health (part C). Aa | Detecting and quantifying endogenous LIN28A protein levels enables qualitative distinction between undifferentiated human induced pluripotent stem cells and differentiated cells. Ab | RNA aptamers can be combined to create an aptamer-based Förster resonance energy transfer (FRET) system for studying RNA folding inside living cells. Ac | A tetracycline-dependent ribozyme switch controls polyQ-huntingtin expression and inclusion body formation in a novel inducible Caenorhabditis elegans polyglutamine Huntington’s disease model. Ba | Cytoplasmic small-molecule concentrations can be monitored with ribozyme switches engineered to respond to specific metabolites. Bb | Metabolite-responsive ribozyme switches integrated into cellular pathways enable reprogramming of networks for dynamic control of metabolic flux. Ca | Insertion of a 6R-folinic acid (6R-FA)-responsive aptamer into microRNA (miRNA) switches enables regulation of T cell proliferation through control of miRNA processing. Cb | CRISPR guide RNA (gRNA) engineered with RNA aptamers that use a strand-displacement mechanism can lead to transcriptional regulation by ‘dead’ CRISPR–Cas9 (dCas9) (REF.). Cc | A ribozyme switch can enable inducible control of anti-vascular endothelial growth factor (anti-VEGF) proteins in a mouse model of wet age-related macular degeneration (AMD). GlcNAc, N-acetylglucosamine.

Fig. 3 |. Novel methods accelerate RNA sensor selection.

Overview of two methods for selection of RNA aptamers or sensors. Both methods begin with a diverse DNA library consisting of 10¹²–10¹⁵ distinct members. a | In the Capture-SELEX (systematic evolution of ligands by exponential enrichment) method^,,, initial sequences consist of a 5′ and 3′ constant region flanking two randomized regions of 10 or 40 nucleotides with a constant docking sequence in the centre. The transcribed library is then hybridized to a biotinylated oligonucleotide via base pairing of the docking sequence. The complexes are immobilized on streptavidin-coated magnetic beads and mixed with the target molecules. RNA sequences that bind the target molecules and undock from the biotinylated oligonucleotides are subsequently eluted from the beads, creating the starting library for the next selection cycle. b | In the DRIVER (de novo rapid in vitro evolution of RNA biosensors) method, libraries are designed based on a modified hammerhead ribozyme (HHRz) from satellite RNA of tobacco ringspot virus (sTRSV) — a small, naturally occurring self-cleaving ribozyme — with the two loops replaced by either a randomized 30–60mer or a randomized 4–8mer. Sequences are mixed with target molecules. Desired RNA biosensor sequences cleave in the absence of the target molecule but bind to the target molecule when present, preventing cleavage. In iterative cycles of negative and positive selection, the RNA sequences are reverse transcribed (txn) and the resultant cDNA of cleaved sequences has a 5′ primer ligated that allows for selective PCR amplification of the sequences corresponding to either the cleaved or uncleaved RNA. The amplified DNA library then serves as the starting library for a new round of DRIVER. Periodically, these libraries can be analysed following quantification by next-generation sequencing (NGS). Part a reprinted with permission from REF, Elsevier.

Fig. 4 |. Emerging mechanisms of RNA processing and cell control for novel RNA devices.

Recent findings have revealed new roles and mechanisms for RNA beyond control of translation through the central dogma, including targeted RNA base editing and post-transcriptional modifications (part A), circular RNA (circRNA) mechanisms (part B) and novel uses of RNA scaffolds (part C). Future RNA devices can incorporate these new mechanisms. Aa | Post-transcriptional modifications are involved in RNA regulation. N⁶-Methy[adenosine (m⁶A) is a post-transcriptional modification affecting mRNA stability and translation. Methyltransferases can be fused to dCas13 (a catalytically inactive CRISPR-associated nuclease) directed by a guide RNA to catalyse the conversion of adenosines to m⁶A, allowing targeted artificial methylation of RNA which could be further utilized. Ab | RNA base editors use naturally occurring and evolved enzymes to modify RNA nucleobases for post-transcriptional gene editing. Current editors utilize deaminases to convert adenosine and cytosine to inosine and uracil, respectively^,,,. Ba | circRNA expression vectors express proteins for longer than equivalent mRNA sequences. Bb | Synthetic circRNAs can be designed as microRNA (miRNA) sponges to quench specific miRNAs and inhibit miRNA-dependent viral replication. Ca | RNA can be repurposed as a scaffold to co-localize enzymes and increase local enzyme concentrations for control of metabolite production. Cb | RNA scaffolds can also be constructed to induce proximity oligomerization of Caspase 8 within the caspase pathway for apoptosis. ADAR, adenosine deaminase acting on RNA. Part Aa is adapted from REF, Springer Nature Limited. Part Cb is adapted from REF, CC-BY 4.0 (https://creativecommons.org/licenses/by/4.0/).