Twister ribozymes as highly versatile expression platforms for artificial riboswitches

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.

Engineering a Functional small RNA Negative Autoregulation Network with Model-guided Design

“…Specifically, synthetic biologists have engineered a diverse array of RNA regulators that can be used to control many of the core processes of gene expression including transcription [2][3][4][5] , translation [6][7][8] and mRNA degradation [9][10][11][12] . In addition to being able to tune the expression of individual genes, these engineered RNA regulators have also been connected together to create synthetic gene networks in the form of logic gates 2,3,5,7,13 , cascades 2,5,[13][14][15][16][17] , single input modules 5,16 feed forward loops 18 , and hybrid feedback controllers 12 .…”

Atom‐Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active‐Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes

“…[1,2] To this end, the three-dimensional architectures of twister, [3][4][5][6] twister sister, [7,8] and pistol ribozymes [9,10] in their pre-catalytic states have been recently revealed by crystallography.T hese provide as olid foundation for mechanistic proposals that await experimental evaluation in solution by structurefunction analysis and targeted atomic mutagenesis.W hile for twister ribozymes,chemical, [4,6,11,12] biochemical, [1][2][3]13] and computational [14] studies have already provided valuable insights into the molecular mechanism of these ribozymes, [15,16] such studies lag behind for the three other new ribozyme classes.Thus far, pre-catalytic structures of the pistol ribozyme have been reported from two groups, [9,10] with good consensus on the overall folding topology and the alignment of catalytic residues at the cleavage site (Figure 1). Our recent crystal structure of ap re-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53-U54 cleavage site.W hile the N1 of G40 is within 3.4 of the modeled G53 2'-OH group that attacks the scissile phosphate, thus suggesting adirect role in general acid-base catalysis,the function of A32 is less clear.W ep resent evidence from atomspecific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis.Bycontrast, the ribose 2'-OH of A32 seems crucial for the proper positioning of G40 through aH-bond network that involves G42 as abridging unit between A32 and G40.…”

“…[1,2] To this end, the three-dimensional architectures of twister, [3][4][5][6] twister sister, [7,8] and pistol ribozymes [9,10] in their pre-catalytic states have been recently revealed by crystallography.T hese provide as olid foundation for mechanistic proposals that await experimental evaluation in solution by structurefunction analysis and targeted atomic mutagenesis.W hile for twister ribozymes,chemical, [4,6,11,12] biochemical, [1][2][3]13] and computational [14] studies have already provided valuable insights into the molecular mechanism of these ribozymes, [15,16] such studies lag behind for the three other new ribozyme classes. [1,2] To this end, the three-dimensional architectures of twister, [3][4][5][6] twister sister, [7,8] and pistol ribozymes [9,10] in their pre-catalytic states have been recently revealed by crystallography.T hese provide as olid foundation for mechanistic proposals that await experimental evaluation in solution by structurefunction analysis and targeted atomic mutagenesis.W hile for twister ribozymes,chemical, [4,6,11,12] biochemical, [1][2][3]13] and computational [14] studies have already provided valuable insights into the molecular mechanism of these ribozymes, …”

A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

“…RNA biosensors have been used as genetically-encoded controllers for a variety of synthetic biology applications 41,42 . Expanding the diversity of ligand-responsive RNA switches can increase the number and complexity of logical operations in biological circuits [43][44][45] . In biomanufacturing, biosensors that can monitor the accumulation of intermediate or product metabolites can be applied to generate screens of selections for improved enzyme activity or pathway flux 15,46 or to implement dynamic feedback control 45 .…”