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Rapid, nondenaturing RNA purification using weak anionexchange fast performance liquid chromatography
LAURA E.EASTON, YOKO SHIBATA, and PETER J. LUKAVSKY Published by Cold Spring Harbor Laboratory Press
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YIWEI DIAO Advance Molecular Biology

Outline

The new role of RNA. Why novel method is needed to purify RNA? The novel method using weak anionexchange via FPLC. Conclusion.

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New role of RNA

In the posttranscriptional regulation of gene expression through RNA interference Many of these (RNAi). regulatory RNA elements conserve the potential to fold into complex structures suggesting that their function is at least partly encodedRNA. Cell 136: 577580. Sharp PA. 2009. The centrality of in their

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http://valelab.ucsf.edu/research/recentresearch/rnaiscreens/rnaiscreens.html http://en.wikipedia.org/wiki/RNAi

A thorough biochemical characterization combined with structural analysis by X-ray crystallography and NMR spectroscopy is imminent if we aim to understand how these RNAs perform their biological functions. RNA can be easily prepared by in vitro transcription from DNA templates using T7 RNA polymerase, but purification of the final RNA product still remains problems.

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Time-consuming Denature RNAs

Batey RT, Kieft JS. 2007. Improved native affinity purification of RNA. RNA 13: 13841389. Lukavsky PJ, Puglisi JD. 2004. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10: 889893.

Novel method: weak anionexchange via FPLC

In vitro transcription of RNA(K10 TLS) from linearized plasmid DNA templates

4 mM each rNTP 70 mg/mL linearized plasmid

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1200 U/mL T7 RNA polymerase 40 mM Tris-HCl (pH 8.1)

Denaturing PAGE analysis of large-scale transcriptions

Kern JA, Davis RH. 1997. Application of solution equilibrium analysis to in vitro RNA transcription. Biotechnol Prog 13: 747756.

Purification of in vitro transcribed RNA

A typical transcription reaction contains several components:

Near-neutral pI (No charge)

the T7 RNA polymerase

A small number of negatively charged phosphate groups

Unincorporated rNTPs small abortive transcripts

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An increased, size-dependent negative charge Highest overall phosphate charge

RNA transcripts (>30 nt)

Grodberg J, Dunn JJ. 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol 170: 12451253.

Equipment
DEAE-sepharose, a weak anion-exchange matrix, was chosen, since it would only require a low salt concentrations to elute the RNA product. AKTA prime FPLC system was chosen a 50 mL superloop
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three 5 mL HiTrap DEAE-sepharose FastFlow columns a single HiPrep 20 mL FF DEAE column

Experiment Procedure

Buffer A (50 mM sodium phosphate [pH 6.5], 150 mM sodium chloride, and 0.2 mM EDTA) Buffer B contains the same components with 2M sodium chloride

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The stopped transcription reaction (1040 mL) is loaded into the 50 mL superloop Collected 10 mL fractions in sterile 15
http://www.genohelix.com/fplctraini

Results

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Elution profile of the 48 nt K10 TLS purified from a 20 mL in vitro transcription reaction using DEAEsepharose chromatography

Denaturing PAGE analysis of the eluted fractions shows that the plasmid DNA template is well separated from the desired RNA product Denaturing SDS PAGE analysis of the eluted fractions 5/3/12 The purified RNA is free of T7 RNA polymerase

Gel filtration of the purified K10 TLS RNA

gray line: UV trace of the crude in vitro transcription of K10 TLS RNA black line: pooled RNA fractions (1721)

Denaturing PAGE analysis of recover rate. Serial dilutions of the crude 20 mL transcription (lane 1, 2 mL; lane 2, 1 mL; lane 3, 0.5 mL; lane 4, 0.25 mL) and the 50 mL pooled, purified RNA fractions 1721 (lane 5, 5 mL; lane 6, 2.5 mL; lane 7, 1.25 mL; lane 8, 0.625 mL)

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An additional benefit of this purification method is that monomeric and multimeric RNA species can be also separated, if their overall charge is significantly different

Denaturing PAGE showed that both peak fractions contained the clean RNA product

Elution profile of the 50 nt hairy SL1 RNA purified from a 20 mL in vitro transcription reaction using DEAE-sepharose chromatography.

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light gray line: UV trace of the crude in vitro transcription black line: pooled monomeric RNA fractions (17 21)

Gel filtration of the purified hairy SL1 RNA

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Renaturation of the oligomeric RNA species by heating and rapid cooling on ice analyzed by gel filtration.

Conclusion
Using this method

Dose not require tedious phenol/chloroform extraction of the T7 RNA polymerase Dose not denature RNA, which is desirable especially for large RNAs Time saving (less than 4h). It is important for biochemical and structural applications Easy to recycle the isotopically labeled rNTPs which are expensive Oligomeric RNA aggregates can be

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References
Grodberg J, Dunn JJ. 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol 170: 1245 1253. Kern JA, Davis RH. 1997. Application of solution equilibrium analysis to in vitro RNA transcription. Biotechnol Prog 13: 747756. Batey RT, Kieft JS. 2007. Improved native affinity purification of RNA. RNA 13: 13841389. Lukavsky PJ, Puglisi JD. 2004. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10: 889893. Kim I, McKenna SA, Viani Puglisi E, Puglisi JD. 2007. Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 13: 289294. Sharp PA. 2009. The centrality of RNA. Cell 136: 577580. http://valelab.ucsf.edu/research/recentresearch/rnaiscreen s/rnaiscreens.html http://en.wikipedia.org/wiki/RNAi

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Thank you!

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