Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography

LAURA E.EASTON, YOKO SHIBATA, and PETER J. LUKAVSKYPublished by Cold Spring Harbor Laboratory Press

YIWEI DIAOAdvance Molecular Biology

Outline

» The new role of RNA.» Why novel method is needed to purify RNA?» The novel method – using weak anion-exchange

via FPLC.» Conclusion.

New role of RNAIn the post-transcriptional regulation of gene expression through RNA interference (RNAi).

Many of these regulatory RNA elements conserve the potential to fold into complex structures suggesting that their function is at least partly encoded in their tertiary fold.Sharp PA. 2009. The centrality of RNA. Cell 136: 577–580.http://valelab.ucsf.edu/research/recentresearch/rnaiscreens/rnaiscreens.htmlhttp://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.

» Time-consuming» Denature RNAs» Limited to small RNAs(<40 nt)» Oligomeric RNA species can not separated from

monomeric speciesBatey RT, Kieft JS. 2007. Improved native affinity purification of RNA. RNA 13: 1384–1389.Lukavsky PJ, Puglisi JD. 2004. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10: 889–893.Kim I, McKenna SA, Viani Puglisi E, Puglisi JD. 2007. Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 13: 289–294.

Novel method: weak anion-exchange via FPLCIn vitro transcription of RNA(K10 TLS)from linearized plasmid DNA templates

4 mM each rNTP 70 mg/mL linearized plasmid 1200 U/mL T7 RNA polymerase 40 mM Tris-HCl (pH 8.1) 1 mM spermidine 5 mM dithiothreito 0.1% Triton-X 100 1 U/mL inorganic pyrophosphatase magnesium chloride.at 37°C

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: 747–756.Lukavsky PJ, Puglisi JD. 2004. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10: 889–893.

» A typical transcription reaction contains several components:

the T7 RNA polymerase

Unincorporated rNTPs small abortive transcripts

RNA transcripts (>30 nt)

large plasmid DNA template

A small number of negatively charged phosphate groups

An increased, size-dependent negative charge

Highest overall phosphate charge

Near-neutral pI (No charge)

Purification of in vitro transcribed RNA

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.

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 three 5 mL HiTrap DEAE-sepharose FastFlow columns a single HiPrep 20 mL FF DEAE column

Equipment

» 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

» The stopped transcription reaction (10–40 mL) is loaded into the 50 mL superloop

» Collected 10 mL fractions in sterile 15 mL plastic tubes

Experiment Procedure

http://www.genohelix.com/fplctraining.htm

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

Results

Denaturing PAGE analysis of the eluted fractions

Denaturing SDS PAGE analysis of the eluted fractions

The purified RNA is free of T7 RNA polymerase

shows that the plasmid DNAtemplate is well separated from the desired RNA product

Gel filtration of the purified K10 TLS RNA Denaturing PAGE analysis of recover rate.

• gray line: UV trace of the crude in vitro transcription of K10 TLS RNA

• black line: pooled RNA fractions (17–21)

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 17–21 (lane 5, 5 mL; lane 6, 2.5 mL; lane 7, 1.25 mL; lane 8, 0.625 mL)

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

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

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

Gel filtration of the purified hairy SL1 RNA

light gray line: UV trace of the crude in vitro transcriptionblack line: pooled monomeric RNA fractions (17–21)gray line: pooled oligomeric RNA fractions (22–28)

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

» 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 separated from the natively folded monomeric RNA product

ConclusionUsing this method…

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: 747–756.Batey RT, Kieft JS. 2007. Improved native affinity purification of RNA. RNA 13: 1384–1389.Lukavsky PJ, Puglisi JD. 2004. Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10: 889–893.Kim I, McKenna SA, Viani Puglisi E, Puglisi JD. 2007. Rapid purification of RNAs using fast performance liquid chromatography (FPLC). RNA 13: 289–294.Sharp PA. 2009. The centrality of RNA. Cell 136: 577–580.http://valelab.ucsf.edu/research/recentresearch/rnaiscreens/rnaiscreens.htmlhttp://en.wikipedia.org/wiki/RNAihttp://www.genohelix.com/fplctraining.htm

Thank you!