Cell-free expression of RNA encoded genes using MS2 replicase

Abstract

RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.

In vitro characterisation of the MS2 RNA polymerase complex reveals novel host factors that modulate leviviral replicase activity

“…All readout constructs are based on designs by Weise et al (20). For experiments using the PURExpress® In Vitro Protein Synthesis Kit (NEB), standard reactions were supplemented with 10 µM DFHBI-1T, MS2rep and F30-Bro(-) or MS2-Bro(+), respectively, as described for the individual experiments: Fluorescence signals were recorded every 60 s over a total of four hours or every 120 s for six hours for experiments involving F30-Bro(+|-) or MS2-Bro(+|-), respectively.…”

“…Next, we sought to explore if the MS2•S1•EF-Ts heterocomplex could be used to initiate transcription of a genuine MS2 template. To this end, we made use of our previously established MS2 RNA polymerase assay for the detection of MS2 replicase activity in recombinant in vitro transcription translation (PURE) systems (20,28). In this assay, a uorescence readout is produced by (+) strand synthesis of the broccoli aptamer from a (-) strand template that is fused with the 3'-end of genomic MS2 (-) strand (F30-Bro(-)), in the presence of the uorogen DFHBI-1T (29) (Fig.…”

“…Moreover, MS2 is used as a surrogate for human pathogenic RNA viruses to study the properties, stability and detectability of RNA viruses under different environmental conditions (7)(8)(9). Apart from their relevance to RNA biochemistry and molecular biology, protein and RNA components of ssRNA coliphages such as Qβ and MS2 serve as attractive platforms for RNA imaging (10)(11)(12), RNA packaging and delivery (13)(14)(15), and molecular evolution and gene expression in cell-free systems (16)(17)(18)(19)(20).…”

“…In particular, a host factor postulated almost 50 years ago (25,26), that is required for RNA replicase activity remains unknown. Here, using our previously established reporter system for MS2 replicase activity in PURE systems (20), we are able to identify new host factors that modulate the activity of the phage enzyme.…”

Applications of phage-derived RNA-based technologies in synthetic biology

“…However, due to its instability and challenging purification [ 96 ], most applications of MS2 phage were focused on RNA-RNA and RNA-protein interactions, such as the RNA imaging mentioned above. A recent study also reported the use of MS2 phage replicase to construct an in-vitro expression system, which provides new ideas for studying the template specificity of MS2 replicase and designing gene circuits with DNA- and RNA-encoded systems [ 98 ]. In addition, there are also reports on the replicases of other types of RNA phages, such as R17 and f2 phage belonging to group I [ 99 , 100 ], GA phage belonging to group II [ 101 ], SP phage belonging to Group IV [ 102 ], and the dsRNA phage ϕ6 belonging to the Cystoviridae family [ 103 ].…”