A general method for rapid and nondenaturing purification of RNAs

Abstract

A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3'-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant HdeltaV ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.

FIGURE 1.

The general scheme for the native purification of the desired sequence (RNA X) using a two-domain affinity tag.

FIGURE 2.

(A) Sequence of the cloning region and affinity tag in pRAV4 (RAV = RNA Affinity Vector). The asterisk denotes the location of the boundary between the RNA of interest and the HδV ribozyme. All unique restriction sites have been denoted in boldface in the vector sequence, and the various functional regions of the vector have been labeled. (B) Sequence of the cloning region and tag of pRAV12. (C) Secondary structure of the RNA affinity tag; the sequence is that of pRAV12. The location of the C75U mutation is boxed.

FIGURE 2.

(A) Sequence of the cloning region and affinity tag in pRAV4 (RAV = RNA Affinity Vector). The asterisk denotes the location of the boundary between the RNA of interest and the HδV ribozyme. All unique restriction sites have been denoted in boldface in the vector sequence, and the various functional regions of the vector have been labeled. (B) Sequence of the cloning region and tag of pRAV12. (C) Secondary structure of the RNA affinity tag; the sequence is that of pRAV12. The location of the C75U mutation is boxed.

FIGURE 2.

(A) Sequence of the cloning region and affinity tag in pRAV4 (RAV = RNA Affinity Vector). The asterisk denotes the location of the boundary between the RNA of interest and the HδV ribozyme. All unique restriction sites have been denoted in boldface in the vector sequence, and the various functional regions of the vector have been labeled. (B) Sequence of the cloning region and tag of pRAV12. (C) Secondary structure of the RNA affinity tag; the sequence is that of pRAV12. The location of the C75U mutation is boxed.

FIGURE 3.

Purification of the T. maritima Ffh M domain (TmaM) as analyzed by a 15% SDS-PAGE gel. (Lane 1) Cells prior to induction with 1 mM IPTG; (lane 2) cells after induction with 1 mM IPTG; (lane 3) supernatant fraction of the cell lysate; (lane 4) fraction of protein eluted from the Ni²⁺-affinity column; (lane 5) protein following cleavage with TEV protease; (lane 6) peak fraction containing TmaM from the SP-Sepharose column. The major band in each lane (except for lane 1) is TmaM.

FIGURE 4.

Test purification of RNA transcribed from the linearized pRAV4 vector. The RNA was body-labeled using [α-³²P]GTP during transcription. An aliquot of the raw transcription reaction is shown on the left, and wash/elution/regeneration fractions are shown. The pure product RNA is indicated.

FIGURE 5.

(A) Crystals of the T. thermophila ΔC209P4–P6 domain RNA that was transcribed and purified using the affinity-tag protocol. (B) Diffraction pattern of crystals showing clear peaks extending to at least 2.87 Å resolution.

FIGURE 5.

(A) Crystals of the T. thermophila ΔC209P4–P6 domain RNA that was transcribed and purified using the affinity-tag protocol. (B) Diffraction pattern of crystals showing clear peaks extending to at least 2.87 Å resolution.