Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides

Abstract

We present a fast and simple protocol for large-scale preparation and purification of RNA oligonucleotides. RNA oligonucleotides are prepared by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates constructed by PCR. In place of denaturing polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography is employed to purify the RNA oligonucleotide from the transcription mixture yielding >99% pure RNA product. In contrast to PAGE-based purification, the gel filtration method does not require denaturation of the RNA oligonucleotide, which is desirable for larger RNAs, and the product is free of low-molecular-weight acrylamide contaminants, which greatly benefits NMR, crystallographic, and other biophysical studies of large RNAs and RNA-protein complexes.

FIGURE 1.

Sequence, design, and PCR construction of DNA templates for in vitro transcription. (A) Synthetic DNA template and design of plasmid DNA template for in vitro transcription of HCV IRES domain IIId (Lukavsky et al. 2000). (B) PCR amplification of DNA template for in vitro transcription of HCV IRES domain IIId (Lukavsky et al. 2001). (C) PCR strategy using overlapping primers for construction of DNA template for in vitro transcription of HCV IRES domain IIId (Lukavsky et al. 2000).

FIGURE 2.

Denaturing PAGE analysis of RNA yield from in vitro transcriptions at different magnesium concentrations. RNA, plasmid DNA and NTPs are visualized by staining with 0.1% toluidine blue.

FIGURE 3.

RNA purification by size-exclusion chromatography. (A) Denaturing PAGE analysis of individual 5-mL fractions collected from size-exclusion chromatography performed on in vitro transcribed RNA. RNA is visualized by staining with 0.1% toluidine blue. (B) Elution profile obtained from the same size-exclusion chromatography. The optical absorbance of each 5-mL fraction was measured at 260 nm.

FIGURE 4.

1D NMR spectra of HCV IRES domain II (Lukavsky et al. 2003). (A) 1D NMR spectrum of HCV IRES domain II purified by denaturing PAGE. The proton resonance lines arising from the acryl-amide oligomers are indicated by arrows. (B) 1D NMR spectrum of HCV IRES domain II purified by size-exclusion chromatography. The arrows in the same positions as in Figure 1A ▶ indicate the absence of acrylamide oligomers.