Rational Design of Versatile RNA-Based Genetic Programs

Rational Design of Versatile RNA-Based Genetic Programs

Abstract

RNA molecules are key regulators of cellular networks in all organisms. Recently, non-coding RNA (ncRNA) regulators have been used in the programming of complex genetic systems. However, it remains a major challenge in the field of RNA synthetic biology to create multiple orthogonally acting ncRNAs and to assemble them into useful regulatory networks. Here we demonstrate a systemic approach from designing ncRNA regulators (parts) to assembling them into higher-order circuits (circuitry). On the parts level, we rationally mutated the natural RNA regulators to create orthogonally acting ncRNAs. Two primary types of ncRNAs that could control either transcription elongation (pT181) or translation initiation (IS10) have been engineered. For the translational ncRNA, we further developed a computer-aided algorithm that predicts the specificity of RNA-RNA interactions and allows us to design more orthogonal ncRNAs. The engineered ncRNAs could also be fused to RNA aptamers as chimeras that could sense small molecules or proteins. We discuss the principles for designing such allosteric chimera molecules. On the circuitry level, we combined multiple ncRNA regulators into higher-order functions. We demonstrate that multiple ncRNAs could form useful circuits that independently control a large number of genes in a single cell, perform logic computation of cellular inputs, and propagate signals along transcriptional cascades. Together, our work demonstrated ncRNAs as useful and powerful genetic tools that can be rationally created in vitro, in vivo, and in silico, and combined into complex circuits useful for therapeutic and manufacturing applications.

Introduction

RNA molecules play key roles in the cellular networks in all organisms (1, 2). Different types of natural RNA regulators have been characterized. These molecules can sense various cellular signals (3, 4), control gene expression (5), and coordinate proteins in complex cellular networks (6). This versatility makes RNAs as promising synthetic tools for the programming of novel genetic systems useful for therapeutic and manufacturing applications (7-10). However, efforts in RNA synthetic biology have been largely limited by two major challenges (5). The first challenge is the scarcity of available RNA regulators with well-defined sensing and regulatory functions. The second one is the absence of design principles that allow efficient and predictable assembly of RNA regulators into networks.

Biomolecular design of orthogonal, versatile, and reliable RNA regulators

To address the first challenge, we developed orthogonal families of bacterial small non-coding RNA (ncRNA) regulators that control target genes independently in vivo. We focused on two primary types of ncRNA regulators that modulate the 5’ cis-regulatory signals encoded on mRNAs. One is the regulator of transcription elongation, which controls RNA polymerase processivity during mRNA biogenesis. One natural example is the antisense RNA system characterized from Staphylococcus aureus pathogenic plasmid pT181 (11). The other is the regulator of translation initiation, which controls ribosome binding site (RBS) accessivity during protein synthesis. A natural example is the antisense RNA system derived from the insertion element IS10 of the Escherichia coli transposon Tn10 (12).

Functional complexity of the system relies on the connections between orthogonally acting genetic components. To create synthetic orthogonal ncRNA-mRNA pairs, we studied how to design novel RNA-RNA interactions. Previous studies have shown that the natural RNA molecules are largely modular. They fold into multiple stem-loop hairpin structures, in which the loop region determines the RNA-RNA interaction, and the stem region provides structural supporting. The loop region usually contains a well-defined RNA motif called YUNR (Y-pyrimidine, R-purine, N-Y or R), which has been reported as a critical element for efficient interaction (11). We thus manipulated this motif and the neighbour nucleotides on the loop region to create new RNA-RNA interactions. We found that this strategy could efficiently disrupt interactions between non-cognate ncRNA-target pairs (13), while maintaining strong interactions between cognate pairs. Furthermore, when we combined the loop mutation strategy with base pair swapping of the stem, we obtained a library of orthogonal pairs almost without crosstalk.

Similar strategies were applied to design orthogonally acting translational RNA regulators based on the natural IS10 system. To modulate specificity, we randomly mutated five nucleotides (nt) on the antisense RNA and the corresponding region of the mRNA, and built a library of 23 mutant pairs. Experimental in vivo fluorescence data of this library were used to train a Partial Least Square Regression (PLSR) model based on thermodynamic considerations. Surprisingly, the regression showed a high correlation between the measured and predicted interactions. This model was then further used to predict new orthogonal pairs in silico, which were verified experimentally. A library of six mutually orthogonal ncRNA-mRNA pairs has been designed using the model (14). Therefore, our results suggest a de novo design method of creating new orthogonal RNA regulators to control protein synthesis.

A large number of trans-acting ncRNA molecules function solely as wires of gene circuits, linking the activity of one genetic element to another. They therefore lack the ability to directly sense other molecular ligands. To design this sensing ability into orthogonal RNA regulators, we fused ncRNAs to RNA aptamers as allosteric chimera molecules (15). RNA aptamers are RNA motifs that could specifically bind to other molecules such as proteins or small molecules. We demonstrated two effective strategies to design allosteric RNA chimeras. In one design, we created a pseudoknot interaction between the loops of the aptamer and ncRNA. The designed pseudoknot could inhibit ncRNA activity in the absence of the ligand (theophylline); but in the presence of the ligand, the RNA aptamer is stabilized by the ligand, eliminating the disruptions from the pseudoknot and restoring the ncRNA regulatory function. In the second design, we introduced a modular linker region between the aptamer and ncRNA, which could flexibly bind to these two modules via base pairing. In the absence of the ligand, the linker region bound to the RNA aptamer and disrupted the ncRNA activity; when the ligand bound to the aptamer, the linker region was released to bind to ncRNA, which restored its function. In vivo fluorescence verified the feasibility of both designs. We applied the strategies to create RNA chimeras that could sense proteins such as the MS2 coat protein or small molecules such as theophylline, and control either transcription or translation. Furthermore, the latter design is modular, and works for different mutants of antisense RNA. We simply changed the nucleotides in the loop region in the RNA chimeras, and the modified chimeras could control new mRNA targets with high specificity.

A RNA-level engineering platform based on synthetic RNA regulators

To address the second challenge, we assembled multiple engineered orthogonal ncRNA regulators into RNA-level genetic circuits. We first constructed three simple but fundamental circuits in vivo using orthogonal transcriptional regulators (16). The first circuit independently controls separate genes using orthogonal regulators in a single cell. The second circuit logically integrates multiple input signals into a tandem cis-regulatory region on the mRNA molecule. Because the presence of each antisense RNA represses gene expression, this circuit represents a NOR logic function, which is the basis for building any other logic types (17). The third circuit is an RNA-only transcriptional cascade. In this circuit, one cis-regulatory element controls the transcription of another orthogonal ncRNA, which controls its own cis-regulatory element. Thus, information can be transmitted and propagated along the synthetic RNA cascade. This is the first reported RNA-based transcriptional cascade, and no natural examples of RNA-only cascade have been reported to date. Since the RNA molecules can be degraded much faster than proteins, we observed extremely fast switching properties from these RNA-level circuits which use short-lived RNAs as intermediate signaling molecules.

To engineer sensory-level circuits, we co-expressed two RNA chimera molecules in the same cell that respond to two different cellular signals (theophylline and MS2 coat protein) and control a single UTR target (15). Presence of any cellular signal activated the cognate RNA chimera molecule, which repressed UTR function and thus gene expression. In this way, we obtained a sensory-level NOR gate.

Summary

To sum, we demonstrate a versatile RNA-level genetic programming platform for encoding novel regulatory functions in organisms. The work uncovered design rules for creating orthogonal libraries of ncRNA regulators that could control transcription or translation, and sense different type of cellular signal inputs. We investigated how to assemble multiple RNA elements into a single transcript regulatory system. Together with technologies for genome-scale modification, our work establishes a foundation for the efficient and predictable engineering of genetic systems based on ncRNA regulators, which provide a novel engineering route to meet demands for new therapies, manufacturing, and the environment.

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