Improved native affinity purification of RNA

Abstract

RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable.

FIGURE 1.

(A) Scheme for the native purification of any desired sequence (“RNA X”) using the new affinity tag. H₆ denotes a 6x histidine tag. (B) Sequence of the RNA expression vector pRAV23 with the relevant restriction sites and elements denoted. The asterisks above the AG step near the NgoMIV site denotes the cleavage site for the glmS ribozyme such that the adenine will be left at the 3′-end of the RNA product. The sequence between the EcoR1 and HindIII sites is inserted into pUC19 to yield pRAV23.

FIGURE 2.

(A) Ethidium bromide stained 12% denaturing polyacrylamide gel of transcription of a 94-nt RNA with the 3′ affinity tag attached in different transcription buffers. (Lane 1) Standard Tris transcription buffer with 20 mM DTT; (lane 2) standard Tris transcription buffer with 10 mM DTT; (lane 3) transription with Na-HEPES (pH 8.0) and 20 mM DTT; (lane 4) transcription with Na-HEPES (pH 8.0) and 10 mM DTT. Product “a” is the full-length product, “b” is the cleaved 3′-tag, and “c” is the 94-nt desired product RNA (which is also the size marker in the “MW” lane). (B) 4%–12% SDS-PAGE analysis of expression and purification of the HMM protein. (Lane 1) Soluble fraction of total cellular lysate; (lane 2) elution from the nickel affinity column; (lane 3) peak from the SP-column. (*) The product protein; molecular size standards are to the right of the gel.

FIGURE 3.

Affinity purification of a 94-nt RNA on small and large scales analyzed on ethidium bromide stained 12% denaturing polyacrylamide gels. (A) Purification of a 100-μL transcription using a QIAGEN Ni-NTA spin column. (Lane Tx) The raw transcription; (lane FT) the column flowthrough; (lanes W1–W3) the three wash steps; (lanes E1,E2) the two 200-μL elutions; (lane S) the imidazole elution. Bands “a”, “b,” and “c” denote the full-length transcript, 3′-tag, and 94-nt product, respectively. (B) Purification of a 3.25-mL transcription using a 3.0-mL Ni-NTA agarose gravity column. Lanes and bands are marked the same as in A, except that there is a third elution step (lane E3).