Extracellular Production of an RNA Aptamer by Ribonuclease-Free Marine Bacteria Harboring Engineered Plasmids: a Proposal for Industrial RNA Drug Production

Abstract

Natural noncoding small RNAs have been shown to be involved in a number of cellular processes as regulators. Using the mechanisms thus elucidated, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutic agents. However, current techniques are too costly for industrial production of these RNAs for use as drugs. Here, we propose a new method for in vivo production of artificial RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. Using engineered plasmids and this bacterium, which produces extracellular nucleic acids in nature, we developed a method for extracellular production of a streptavidin RNA aptamer. As the bacterium does not produce any RNases in the culture medium, at least within the cultivation period tested, the designed RNA itself is produced and retained in the culture medium of the bacterium without any specific mechanism for protection against degradation by nucleases. Here, we report that the streptavidin RNA aptamer is produced in the culture medium and retains its specific function. This is the first demonstration of extracellular production of a functional artificial RNA in vivo, which will pave the way for inexpensive production of RNA drugs.Recent studies have indicated that many small RNAs play key roles in the regulation of gene expression and that higherorder structures in RNA sequences, such as riboswitches or ribozymes, act as regulators of mRNA expression (3,4,17). In addition to these natural RNA functions, artificial small interfering RNAs (siRNAs), ribozymes, and RNA aptamers are also expected to be potential candidates for RNA therapeutics (10,20). An RNA aptamer has already been developed as an RNA drug for the inhibition of macular degradation by specifically targeting the vascular endothelial growth factor (18). In both basic studies of RNA and RNA drug production, efficient methods for preparation of homogeneous RNA molecules are very important. In the case of RNA drug production, economically efficient methods for large-scale production are required.At present, the most reliable methods for preparation of homogeneous RNAs are in vitro transcription using T7 RNA polymerase (15) and chemical synthesis (12). These methods, however, are not suitable for preparation in large quantities because they are both costly and labor intensive. For industrial production of RNAs, in vivo production using microorganisms is thought to be the most suitable method. Recently, Ponchon and Dardel reported in vivo production of recombinant RNAs using Escherichia coli (19). They proposed a system called a "tRNA scaffold" in which the product RNA is designed to be included in a tRNA structure to obtain a homogeneous RNA product. Their product RNA is produced efficiently in homogeneous form, because the in vivo transcript containing the product sequence is processed as a tRNA by the cellular processing system, although the product contains the flanking sequences of tRNA on both sides (19). We have reported...

Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium <i>Rhodovulum sulfidophilum</i>

“…Previously, we developed a method for extracellular production of artificially designed, functional RNAs (RNA aptamers and short hairpin RNAs) in the culture medium using R. sulfidophilum (Kikuchi, 2010;Kikuchi et al, 2010;Nagao et al, 2014;Suzuki et al, 2010Suzuki et al, , 2011Umekage et al, 2012). This method has been proposed for the industrial production of RNA medicines.…”

“…Using this property, we developed a method for the extracellular production of homogeneous recombinant RNA molecules, such as RNA aptamers (reviewed by Kikuchi, 2010;Suzuki et al, 2010Suzuki et al, , 2011Umekage et al, 2012) and short hairpin RNAs (Nagao et al, 2014) by the introduction of an appropriate RNA expression plasmid into the cell. This approach has been proposed as a possible method in industrial technology to produce RNA medicines in the future (Kikuchi, 2010;Kikuchi et al, 2010).…”

New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli

“…20 On the other hand, the production of artificial RNAs in the marine phototrophic bacterium Rhodovolum sulfidophilum (R. sulfidophilum) was first achieved in 2010 by Suzuki and colleagues. 21 In particular, as R. sulfidophilum does not accumulate any RNases in the culture medium, its ability to produce extracellular nucleic acids 21 opens new perspectives in the RNA recombinant technology once their stability is greatly enhanced. To achieve successful recombinant RNA expression in E. coli, researchers usually use a strategy of "camouflage" of the RNA of interest within a stable RNA scaffold 22 that may be achieved using tRNA 11,17,22 or rRNA.…”

“…14 On the other hand, using R. sulfidophilum as the expression host, a similar strategy to the first used by Suzuki and colleagues in 2010 21 was also applied by our research group 7 where the target RNA is flanked by two hammerhead ribozyme sequences that possess self-cleavage activities leading to the releasing of the mature recombinant target RNA. Another important issue for evaluating the therapeutic effect of miRNAs is the target sequence cloned into the plasmid.…”

Current progress on microRNAs‐based therapeutics in neurodegenerative diseases

“…More recently, our research group applied an innovative recombinant bacteria, Rhodovulum sulfidophilum , as the expression host to produce stable recombinant pre‐miRNA (human pre‐miR‐29b) using a similar strategy to the previous one employed by Suzuki et al in 2010 to produce RNA aptamers . In this strategy, the target pre‐miRNA is flanked by two hammerhead ribozyme sequences that possess self‐cleavage activity leading to the release of the recombinant target RNA .…”