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A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

Chen QX, Wang WP, Zeng S, Urayama S, Yu AM - Nucleic Acids Res. (2015)

Bottom Line: We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a).Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture.These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Medicine, School of Medicine, UC Davis, Sacramento, CA 95817, USA Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.

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OnRS-carried siRNA is effective for RNAi in vitro and in vivo. GFP fluorescence intensity was sharply reduced in ES-2/GFP cells in vitro at 72 h after transfected with OnRS/GFP-siRNA (a), which was associated with (b) 70–80% lower GFP mRNA levels and (c) 1000-fold higher GFP siRNA levels. Following i.v. administration of OnRS/GFP-siRNA, hepatic GFP fluorescence was significantly suppressed in the GFP-transgenic mouse models in vivo, as demonstrated by microscopic examination of (d) non-fixed and (e) fixed liver slices, as well as (f) RT-qPCR analysis of hepatic GFP mRNA levels. Fixed liver slices were stained with DAPI, and GFP fluorescence and DAPI-stained nuclei (blue) images were merged together (e). Control ES-2/GFP cells (N = 3 per group) or GFP-transgenic mice (N = 3–4 per group) were treated with the same doses of OnRS/Neg. Values are mean ± SD. *P < 0.01, compared with OnRS/Neg treatment.
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Figure 4: OnRS-carried siRNA is effective for RNAi in vitro and in vivo. GFP fluorescence intensity was sharply reduced in ES-2/GFP cells in vitro at 72 h after transfected with OnRS/GFP-siRNA (a), which was associated with (b) 70–80% lower GFP mRNA levels and (c) 1000-fold higher GFP siRNA levels. Following i.v. administration of OnRS/GFP-siRNA, hepatic GFP fluorescence was significantly suppressed in the GFP-transgenic mouse models in vivo, as demonstrated by microscopic examination of (d) non-fixed and (e) fixed liver slices, as well as (f) RT-qPCR analysis of hepatic GFP mRNA levels. Fixed liver slices were stained with DAPI, and GFP fluorescence and DAPI-stained nuclei (blue) images were merged together (e). Control ES-2/GFP cells (N = 3 per group) or GFP-transgenic mice (N = 3–4 per group) were treated with the same doses of OnRS/Neg. Values are mean ± SD. *P < 0.01, compared with OnRS/Neg treatment.

Mentions: We also assessed the effectiveness of OnRS-carried GFP siRNA using GFP-expressing ES-2 cells and GFP-transgenic mouse models. In ES-2/GFP cells, OnRS/GFP-siRNA significantly suppressed the GFP fluorescence intensity and mRNA levels at 72 h post-transfection (Figure 4a and b), which was associated with 3 orders of magnitude increase in GFP siRNA levels (Figure 4c). We then treated GFP-transgenic mice (24) with in vivo-jetPEI-formulated OnRS/GFP-siRNA. Compared to the GFP-transgenic mice treated with the same doses of in vivo-jetPEI-formulated OnRS/Neg, there was a remarkable reduction of hepatic GFP fluorescence intensity (Figure 4d and e) and mRNA levels (Figure 4f) in GFP-transgenic mice treated with OnRS/GFP-siRNA, which was linked to an over 3000-fold increase in GFP siRNA levels. These data indicate that chimeric GFP-siRNAs produced on large scale using the OnRS cargo are effective agents for in vitro and in vivo RNAi applications.


A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications.

Chen QX, Wang WP, Zeng S, Urayama S, Yu AM - Nucleic Acids Res. (2015)

OnRS-carried siRNA is effective for RNAi in vitro and in vivo. GFP fluorescence intensity was sharply reduced in ES-2/GFP cells in vitro at 72 h after transfected with OnRS/GFP-siRNA (a), which was associated with (b) 70–80% lower GFP mRNA levels and (c) 1000-fold higher GFP siRNA levels. Following i.v. administration of OnRS/GFP-siRNA, hepatic GFP fluorescence was significantly suppressed in the GFP-transgenic mouse models in vivo, as demonstrated by microscopic examination of (d) non-fixed and (e) fixed liver slices, as well as (f) RT-qPCR analysis of hepatic GFP mRNA levels. Fixed liver slices were stained with DAPI, and GFP fluorescence and DAPI-stained nuclei (blue) images were merged together (e). Control ES-2/GFP cells (N = 3 per group) or GFP-transgenic mice (N = 3–4 per group) were treated with the same doses of OnRS/Neg. Values are mean ± SD. *P < 0.01, compared with OnRS/Neg treatment.
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Related In: Results  -  Collection

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Figure 4: OnRS-carried siRNA is effective for RNAi in vitro and in vivo. GFP fluorescence intensity was sharply reduced in ES-2/GFP cells in vitro at 72 h after transfected with OnRS/GFP-siRNA (a), which was associated with (b) 70–80% lower GFP mRNA levels and (c) 1000-fold higher GFP siRNA levels. Following i.v. administration of OnRS/GFP-siRNA, hepatic GFP fluorescence was significantly suppressed in the GFP-transgenic mouse models in vivo, as demonstrated by microscopic examination of (d) non-fixed and (e) fixed liver slices, as well as (f) RT-qPCR analysis of hepatic GFP mRNA levels. Fixed liver slices were stained with DAPI, and GFP fluorescence and DAPI-stained nuclei (blue) images were merged together (e). Control ES-2/GFP cells (N = 3 per group) or GFP-transgenic mice (N = 3–4 per group) were treated with the same doses of OnRS/Neg. Values are mean ± SD. *P < 0.01, compared with OnRS/Neg treatment.
Mentions: We also assessed the effectiveness of OnRS-carried GFP siRNA using GFP-expressing ES-2 cells and GFP-transgenic mouse models. In ES-2/GFP cells, OnRS/GFP-siRNA significantly suppressed the GFP fluorescence intensity and mRNA levels at 72 h post-transfection (Figure 4a and b), which was associated with 3 orders of magnitude increase in GFP siRNA levels (Figure 4c). We then treated GFP-transgenic mice (24) with in vivo-jetPEI-formulated OnRS/GFP-siRNA. Compared to the GFP-transgenic mice treated with the same doses of in vivo-jetPEI-formulated OnRS/Neg, there was a remarkable reduction of hepatic GFP fluorescence intensity (Figure 4d and e) and mRNA levels (Figure 4f) in GFP-transgenic mice treated with OnRS/GFP-siRNA, which was linked to an over 3000-fold increase in GFP siRNA levels. These data indicate that chimeric GFP-siRNAs produced on large scale using the OnRS cargo are effective agents for in vitro and in vivo RNAi applications.

Bottom Line: We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a).Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture.These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Medicine, School of Medicine, UC Davis, Sacramento, CA 95817, USA Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.

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Related in: MedlinePlus