Synthetic RNA-based logic computation in mammalian cells

Satoshi Matsuura,Yoshihiko Fujita,Yi Kuang,Hiroki Ono,Hirohide Saito,Shunsuke Kawasaki

doi:10.1038/s41467-018-07181-2

Abstract

Synthetic biological circuits are designed to regulate gene expressions to control cell function. To date, these circuits often use DNA-delivery methods, which may lead to random genomic integration. To lower this risk, an all RNA system, in which the circuit and delivery method are constituted of RNA components, is preferred. However, the construction of complexed circuits using RNA-delivered devices in living cells has remained a challenge. Here we show synthetic mRNA-delivered circuits with RNA-binding proteins for logic computation in mammalian cells. We create a set of logic circuits (AND, OR, NAND, NOR, and XOR gates) using microRNA (miRNA)- and protein-responsive mRNAs as decision-making controllers that are used to express transgenes in response to intracellular inputs. Importantly, we demonstrate that an apoptosis-regulatory AND gate that senses two miRNAs can selectively eliminate target cells. Thus, our synthetic RNA circuits with logic operation could provide a powerful tool for future therapeutic applications.

Highlights

To reduce off-target effects in non-target cells, it is important to produce desired outputs dependent on the cell state
The circuit topology of this device consists of two types of modRNAs (Fig. 1b); one is an L7Ae-coding mRNA with four miRNA target sites that are completely complementary to the mature miRNA within the 3′ untranslated region (3′-UTR), and the other is an output-gene-coding mRNA with a Kt motif within the 5′-UTR
We refer to this device as L7-4xTX, where 4x represents the number of miRNA target sites, TX represents target sites to the specific miRNA, and the position of TX in the device name represents the location of the target site in the device (i.e., 5′-UTR or 3′-UTR)

Summary

Introduction

To reduce off-target effects in non-target cells, it is important to produce desired outputs dependent on the cell state. The miRNA expression profile is related to important biological processes, including development, cancer, and cell reprogramming, and can be used to classify the cell state5,19–21 These properties suggest that miRNA-responsive, synthetic circuits could provide useful tools for future therapeutic applications. Synthetic circuits using modRNAs that encode RNA binding proteins (RBPs) have been constructed in mammalian cells, complex synthetic RNA-delivered circuits that can detect multiple miRNAs and regulate output protein through logic computation have not been demonstrated. We aimed to design synthetic RNA-delivered logic circuits that function in mammalian cells by improving the performance of miRNA- and protein-responsive modRNAs. In this study, we construct a set of RNA-based logic circuits with RBPs that detect multiple miRNA inputs and regulate the output protein expression (Fig. 1a). We selectively control cell-death pathways between target and non-target cells by connecting a 2-input AND gate with apoptotic regulatory circuits

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Conclusion