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.

FIG. 1.

RNase-free culture medium of R. sulfidophilum. The supernatant of R. sulfidophilum DSM 1374^T or E. coli JM 109 culture medium was incubated with synthetic tRNA substrate (+ Synthetic tRNA) for 30 min or 90 min at 37°C as described in Materials and Methods. The reaction mixtures were analyzed by 10% denaturing PAGE. − Synthetic tRNA, no addition of the substrate to the reaction mixture; R-culture and E-culture, R. sulfidophilum culture medium and E. coli culture medium, respectively; nt, nucleotides. The cultivation time (17, 16, 27, 42, 65, or 87 h) and the reaction time (30 or 90 min) are indicated. RNase A represents the reaction mixture with RNase A instead of culture supernatant as a positive control.

FIG. 2.

Construction of plasmids pHSP1 and pHSR1 and nucleotide sequence and secondary-structure model of the RNA expressed. (a) Organization of RNA aptamer (streptavidin RNA aptamer) expression sites designed in the plasmids. The plasmids pHSP1 and pHSR1 contained the puf promoter and the rrn promoter, respectively. (b) Predicted secondary structure of the expressed RNA. The streptavidin RNA aptamer with flanking hammerhead ribozyme sequences is shown. The self-cleavage sites expected in hammerhead ribozyme reactions are indicated by small arrows. 101N and 126N indicate the predicted additional lengths expressed from the plasmid pHSR1 (rrn promoter). The EcoRI and HindIII sites used for cloning (see Materials and Methods) are indicated.

FIG. 3.

Production and release of the streptavidin RNA aptamer into the culture medium. (a) Detection of the streptavidin RNA aptamer sequence. Using streptavidin RNA aptamer-specific primers, the aptamer sequences were amplified by RT-PCR from intracellular (in) and extracellular (ex) RNA preparations from R. sulfidophilum harboring pHSP1 in the early stationary phase of growth. N and pHSP1 represent the RNA preparations from R. sulfidophilum DSM 1374^T only (no plasmid) and the bacterium transformed with pHSP1, respectively. C indicates a positive control using the in vitro transcript of the streptavidin RNA aptamer as a template for RT-PCR. (b and c) Extracellular production of the streptavidin RNA aptamer. Time courses of extracellular production of the RNA aptamer from the bacteria harboring pHSP1 (b) and pHSR1 (c) are shown. The amounts of RNA aptamers were measured by quantitative RT-PCR as described in Materials and Methods. Closed circles, cell growth (optical densities at 600 nm [OD₆₀₀]); open circles, amounts of extracellular streptavidin RNA (exSA) aptamer (ng/liter culture). (d) Northern blotting analysis of the streptavidin RNA aptamer produced. To confirm the size of the RNA, Northern analysis of extracellular-RNA preparations was performed using a cDNA probe for the streptavidin RNA aptamer. C indicates a positive control using the in vitro transcript of the streptavidin RNA aptamer. Clear, sharp bands are shown. This aptamer has a size of 80 nucleotides and can be used as an accurate size marker. N and pHSR1 represent the extracellular RNA preparations (exRNA) from R. sulfidophilum DSM 1374^T only (no plasmid) and the bacterium transformed with pHSR1, respectively; refold indicates additional incubation to enhance self-cleavage (see the text).

FIG. 4.

Electrophoretic mobility shift assay of the product. To confirm the aptamer function of the extracellularly produced RNA, an electrophoretic mobility shift assay was performed. Partially purified and radiolabeled extracellular RNA preparations (ex) were incubated with 1 μM streptavidin in the presence (lanes 3 and 6) or absence (lanes 1, 2, 4, and 5) of 3.3 mM biotin and analyzed by native PAGE. An autoradiogram of the gel is shown. C indicates a positive control using the in vitro transcript of the streptavidin RNA aptamer. The positions of free and complex RNAs are indicated.