Aptazyme-Mediated Regulation of 16S Ribosomal RNA

Abstract

Developing artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria.

A general design strategy for protein-responsive riboswitches in mammalian cells

“…However, protein-responsive ribozymes have been validated only in vitro and could not be used to control target-gene expression in living cells 31,32 . Ligandcontrolled ribozymes were also successfully used to modulate the activity of other functional RNAs such as tRNA 33 , rRNA 34 and short hairpin RNA 35 , as well as for the control of T-cell proliferation 36 and transgene expression in oncolytic viruses 37 . Nevertheless, the design of trigger-responsive ribozymes remains challenging because the integration of aptamers into ribozymes often impairs critical tertiary interloop structures, which results in moderate switching capacities 38,39 .…”

Thermozymes

“…So far, aptamers binding several ligands were combined with HHRs functioning as expression platforms in order to control mRNAs, tRNAs, 16S rRNA as well as RNAi in organisms as diverse as bacteria and mammalian cells. 11,15,16,21,[23][24][25]41 The present work shows that a physical stimulus such as a change in temperature can also be used in order to regulate gene expression via a hammerhead-mediated mRNA cleavage reaction. Importantly, most natural RNAT and all previously engineered RNAT are composed of secondary structures that mask the SD sequence.…”

“…[18][19][20] In our opinion, ribozyme-based devices have the advantage of almost universal applicability for controlling RNA functions. Apart from regulating mRNA translation in bacteria and mRNA integrity in mammalian cells, 21,22 they can be utilized in order to control the activity of tRNAs, 23 16S rRNA, 24 as well as RNAi in mammalia. 21 In addition, they can be combined in a modular fashion in order to yield two-input Boolean logic operators.…”

RNA-Based Therapeutics: Current Progress and Future Prospects

“…Moreover, ribozymes are amenable to in vitro selection and directed evolution to generate improved properties and new functions for therapeutic and diagnostic reagents. Ribozymes can be engineered to be allosterically activated by effector molecules, which has led to the development of artificial “riboswitches” as biosensors and synthetic biological tools [74, 75]. There are numerous types of ribozymes in biology, but the most common ribozyme therapeutics are derived from either “hammerhead” or “hairpin/paperclip” motifs.…”