Multi-arm RNA junctions encoding molecular logic unconstrained by input sequence for versatile cell-free diagnostics

Duo Ma,Abraham Campos-Romero,Soma Chaudhary,Zachary M Ticktin,Jonathan Alcantar-Fernandez,Anli A Tang,Kaiyue Wu,Zhaoqing Yan,José L Moreno-Camacho,Alexander A Green,Yuexin Li

doi:10.1038/s41551-022-00857-7

Abstract

Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. Here we show that the sequence constraints can be substantially reduced by appropriately encoded multi-arm junctions of single-stranded RNA structures. To conditionally activate RNA translation, we integrated multi-arm junctions, self-assembled upstream of a regulated gene and designed to unfold sequentially in response to different RNA inputs, with motifs of loop-initiated RNA activators that function independently of the sequence of the input RNAs and that reduce interference with the output gene. We used the integrated RNA system and sequence-independent input RNAs to execute two-input and three-input OR and AND logic in Escherichia coli, and designed paper-based cell-free colourimetric assays that accurately identified two human immunodeficiency virus (HIV) subtypes (by executing OR logic) in amplified synthetic HIV RNA as well as severe acute respiratory syndrome coronavirus-2 (via two-input AND logic) in amplified RNA from saliva samples. The sequence-independent molecular logic enabled by the integration of multi-arm junction RNAs with motifs for loop-initiated RNA activators may be broadly applicable in biotechnology.

Highlights

Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits
We found that these clamp sequence changes did not impact the OFF-state signal of the loop-initiated RNA activator (LIRA) (Extended Data Fig. 2c), but they did cause variations in ON-state expression levels ranging from 40% to 230% of the parent LIRA (Extended Data Fig. 2d)
We found that all four mRNAs could be readily detected using the LIRAs and provided ON/OFF green fluorescent protein (GFP) levels ranging from 22- to 38-fold (Fig. 2e)

Summary

Introduction

Applications of RNA-based molecular logic have been hampered by sequence constraints imposed on the input and output of the circuits. The structural diversity of RNA has been harnessed in RNA nanotechnology to generate a variety of RNA-based nanostructures with complex geometries through self-assembly[20–22] These structures are assembled from molecular building blocks featuring hairpins, multi-arm junctions, and other structural elements programmed to fold into prescribed structures through combinations of dangling end, kissing loop, and crossover interactions[23–27]. Taking concepts from RNA nanotechnology and RNA-based regulation of gene expression[33,34], recent years have seen the development of self-assembly-driven molecular computing systems that operate in living cells and exploit the combined interactions of multiple carefully designed synthetic RNAs11,35–37 Such ribocomputing devices act by modulating gene expression in response to specified combinations of input RNAs and take advantage of the predictability of RNA–RNA interactions to enable effective computer-based design. We describe a strategy for implementing molecular logic that exploits multi-arm junction RNA structures to regulate gene

Methods

Results

Conclusion