Limits...
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.

Show MeSH

Related in: MedlinePlus

High-yield production of functional RNA aptamers in bacteria using OnRS. (a) Representative design of OnRS/aptamer forms where RNA aptamer was inserted at the 5′ or 3′ end of hsa-mir-34a. The heat color gradation indicates the base-pairing probability from 0 to 1. (b) A consistent high-level expression of OnRS-carried MGA in E. coli, i.e. over 50% of OnRS/MGA5 and OnRS/MGA3 in total RNAs. (c) Representative FPLC traces of OnRS/MGA5 during FPLC purification. Insert is urea–PAGE analysis of the collected RNA fractions (1, 2 and 3) eluted at 10.6 min. (d) Binding to OnRS/MGA5 and OnRS/MGA3 led to a shift of the wavelength of MG maximum absorbance from 618 to 630 nm. The same shift was observed when FPLC-purified OnRS/MGA and total RNAs isolated from OnRS/MGA-expressing bacteria were used. The sephadex aptamer (OnRS/Seph) and corresponding total RNAs were used as additional controls. (e) Strong and selective fluorescence was shown when MG bound to OnRS/MGA5 or OnRS/MGA3. The same results were obtained when using FPLC-purified OnRS/MGA and OnRS/MGA-containing total RNAs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402540&req=5

Figure 5: High-yield production of functional RNA aptamers in bacteria using OnRS. (a) Representative design of OnRS/aptamer forms where RNA aptamer was inserted at the 5′ or 3′ end of hsa-mir-34a. The heat color gradation indicates the base-pairing probability from 0 to 1. (b) A consistent high-level expression of OnRS-carried MGA in E. coli, i.e. over 50% of OnRS/MGA5 and OnRS/MGA3 in total RNAs. (c) Representative FPLC traces of OnRS/MGA5 during FPLC purification. Insert is urea–PAGE analysis of the collected RNA fractions (1, 2 and 3) eluted at 10.6 min. (d) Binding to OnRS/MGA5 and OnRS/MGA3 led to a shift of the wavelength of MG maximum absorbance from 618 to 630 nm. The same shift was observed when FPLC-purified OnRS/MGA and total RNAs isolated from OnRS/MGA-expressing bacteria were used. The sephadex aptamer (OnRS/Seph) and corresponding total RNAs were used as additional controls. (e) Strong and selective fluorescence was shown when MG bound to OnRS/MGA5 or OnRS/MGA3. The same results were obtained when using FPLC-purified OnRS/MGA and OnRS/MGA-containing total RNAs.

Mentions: Encouraged by these findings, we further challenged the potential applications of OnRS to the production of functional RNA aptamers. A malachite green aptamer (MGA) (9) was chosen as a model aptamer and inserted at the 5′ and 3′ of miR-34a to offer OnRS/MGA5 and OnRS/MGA3, respectively (Figure 5a). Both chimeras were revealed to be expressed at surprisingly high levels in bacteria, i.e. over 50% of OnRS/MGA in total RNAs (Figure 5b). Thus, we could use FPLC (Figure 5c) to easily purify 5–6 mg OnRS/MGA from 15 to 20 mg total RNAs isolated from 0.5 l bacterial culture at all times.


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)

High-yield production of functional RNA aptamers in bacteria using OnRS. (a) Representative design of OnRS/aptamer forms where RNA aptamer was inserted at the 5′ or 3′ end of hsa-mir-34a. The heat color gradation indicates the base-pairing probability from 0 to 1. (b) A consistent high-level expression of OnRS-carried MGA in E. coli, i.e. over 50% of OnRS/MGA5 and OnRS/MGA3 in total RNAs. (c) Representative FPLC traces of OnRS/MGA5 during FPLC purification. Insert is urea–PAGE analysis of the collected RNA fractions (1, 2 and 3) eluted at 10.6 min. (d) Binding to OnRS/MGA5 and OnRS/MGA3 led to a shift of the wavelength of MG maximum absorbance from 618 to 630 nm. The same shift was observed when FPLC-purified OnRS/MGA and total RNAs isolated from OnRS/MGA-expressing bacteria were used. The sephadex aptamer (OnRS/Seph) and corresponding total RNAs were used as additional controls. (e) Strong and selective fluorescence was shown when MG bound to OnRS/MGA5 or OnRS/MGA3. The same results were obtained when using FPLC-purified OnRS/MGA and OnRS/MGA-containing total RNAs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402540&req=5

Figure 5: High-yield production of functional RNA aptamers in bacteria using OnRS. (a) Representative design of OnRS/aptamer forms where RNA aptamer was inserted at the 5′ or 3′ end of hsa-mir-34a. The heat color gradation indicates the base-pairing probability from 0 to 1. (b) A consistent high-level expression of OnRS-carried MGA in E. coli, i.e. over 50% of OnRS/MGA5 and OnRS/MGA3 in total RNAs. (c) Representative FPLC traces of OnRS/MGA5 during FPLC purification. Insert is urea–PAGE analysis of the collected RNA fractions (1, 2 and 3) eluted at 10.6 min. (d) Binding to OnRS/MGA5 and OnRS/MGA3 led to a shift of the wavelength of MG maximum absorbance from 618 to 630 nm. The same shift was observed when FPLC-purified OnRS/MGA and total RNAs isolated from OnRS/MGA-expressing bacteria were used. The sephadex aptamer (OnRS/Seph) and corresponding total RNAs were used as additional controls. (e) Strong and selective fluorescence was shown when MG bound to OnRS/MGA5 or OnRS/MGA3. The same results were obtained when using FPLC-purified OnRS/MGA and OnRS/MGA-containing total RNAs.
Mentions: Encouraged by these findings, we further challenged the potential applications of OnRS to the production of functional RNA aptamers. A malachite green aptamer (MGA) (9) was chosen as a model aptamer and inserted at the 5′ and 3′ of miR-34a to offer OnRS/MGA5 and OnRS/MGA3, respectively (Figure 5a). Both chimeras were revealed to be expressed at surprisingly high levels in bacteria, i.e. over 50% of OnRS/MGA in total RNAs (Figure 5b). Thus, we could use FPLC (Figure 5c) to easily purify 5–6 mg OnRS/MGA from 15 to 20 mg total RNAs isolated from 0.5 l bacterial culture at all times.

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.

Show MeSH
Related in: MedlinePlus