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

• Published in: Applied and Environmental Microbiology 2010-02, Vol.76 (3), p.786-793
• Main Authors: Suzuki, Hiromichi, Ando, Tomoaki, Umekage, So, Tanaka, Terumichi, Kikuchi, Yo
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide - biosynthesis; Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - metabolism; Bacteria; Base Sequence; Biological and medical sciences; Biotechnology; Cells; Cloning, Molecular; Culture Media - metabolism; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Endoribonucleases - genetics; Endoribonucleases - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Industrial Microbiology; Microbiology; Molecular Sequence Data; Nucleic Acid Conformation; Plasmids - genetics; Rhodovulum - enzymology; Rhodovulum - genetics; Rhodovulum - metabolism; Rhodovulum sulfidophilum; Ribonucleases - genetics; Ribonucleases - metabolism; Ribonucleic acid; Ribozymes; RNA; RNA - biosynthesis; RNA - genetics; RNA - metabolism; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Ribosomal, 16S - genetics; RNA, Ribosomal, 16S - metabolism; Sequence Analysis, RNA; Streptavidin - metabolism; Water Microbiology
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.01971-09

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.