In vitro and in vivo production and purification of circular RNA aptamer

Umekage, So; Kikuchi, Yo

doi:10.1016/j.jbiotec.2008.12.012

Cited by 52 publications

(43 citation statements)

References 34 publications

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“…Moreover, the culture volume can be scaled up easily, which is necessary for large-scale industrial preparation. In vivo expression and preparation of artificial RNAs have been reported previously (19,26). We developed a method for in vivo production of circular RNAs using E. coli (26).…”

Section: Discussionmentioning

confidence: 99%

“…In vivo expression and preparation of artificial RNAs have been reported previously (19,26). We developed a method for in vivo production of circular RNAs using E. coli (26). As the RNAs produced by this method are circular molecules, the products are stably maintained in the cells, because such circular molecules are resistant to cellular exonucleases.…”

Section: Discussionmentioning

confidence: 99%

“…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 the in vivo production of a circular RNA aptamer (26). Circularization of the RNA product was accomplished in E. coli by selfsplicing permuted intron-exon sequences derived from the T4 bacteriophage gene td.…”

mentioning

confidence: 99%

“…Circularization of the RNA product was accomplished in E. coli by selfsplicing permuted intron-exon sequences derived from the T4 bacteriophage gene td. The circular RNA was stably produced in vivo because of its resistance to exonucleases (26). In both cases, however, modifications of RNA products, such as the tRNA scaffold or circularization, are necessary to obtain the product efficiently.…”

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confidence: 99%

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

Suzuki

Ando

Umekage

et al. 2010

Appl Environ Microbiol

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Suzuki

Ando

Umekage

et al. 2010

Appl Environ Microbiol

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“…Identification of circular RNAs by 2D electrophoresis is based on the fact that the migration of linear and circular nucleic acids is distinctly dependent on the gel pore size (Sigurdsson and Eckstein 1996;Umekage and Kikuchi 2009). To enrich the samples with linear RNAs for better identification of the circular species, a commercially available RNA size standard (RiboRuler low-range RNA ladder; Fermentas) being composed exclusively of linear RNAs was added.…”

Section: Two-dimensional Electrophoresismentioning

confidence: 99%

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

Petkovic

Badelt

Block

et al. 2015

RNA

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Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and selfconcatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions.

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Tactics targeting circular mRNA biosynthesis

Chen

Wang

2023

Biotech & Bioengineering

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Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self‐splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20‐bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.

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In vitro and in vivo production and purification of circular RNA aptamer

Cited by 52 publications

References 34 publications

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

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

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

Tactics targeting circular mRNA biosynthesis

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