Twister ribozymes as highly versatile expression platforms for artificial riboswitches.

Michele Felletti,Jörg S Hartig,Lena A Wurmthaler,Julia Stifel,Sophie Geiger

doi:10.1038/ncomms12834

Abstract

The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class.

Highlights

Background enhanced green fluorescent protein (eGFP) Act Inact Act InactAct Inact library was screened directly in yeast for differential expression of the reporter gene in the presence and in the absence of neomycin in yeast culture medium
We show that the twister ribozyme is an extraordinarily versatile expression platform that allows the connection of three different aptamer domains at two independent positions (P1 and P5), generating a variety of one-input RNA switches that can act as efficient synthetic genetic regulators in both E. coli and yeast
Using a design that is mimicking the situation observed in some naturally occurring twister ribozymes where multiple optional domains are directly connected to the catalytic core in position P1 and P5, we developed a series of compact two-input riboswitches that sense and respond to two small molecular signals at once (theophylline and thiamin pyrophosphate (TPP)) and that behave as Boolean logic operators in E. coli

Summary

Introduction

Background eGFP Act Inact Act InactAct Inact library was screened directly in yeast for differential expression of the reporter gene in the presence and in the absence of neomycin in yeast culture medium (for details see Supplementary Note 4). The full sequences of all the one-input twister-based riboswitches in E. coli and yeast are shown in the Supplementary Table 4. The results described so far demonstrate that the twister ribozyme scaffold offers at least two independent attachment sites for ligand-sensing aptamer domains. This opens up the possibility of constructing compact two-input genetic switches that sense and respond to two small molecular signals at once. We started from the P1- or P5-based theophylline and TPP riboswitches and sampled libraries of randomized communication modules in the second position with the second aptamer domain attached (Fig. 3, Supplementary Fig. 12 and Supplementary Note 5).

Methods

Results

Conclusion