High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme

Abstract

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme

“…They were visualized by UV shadowing, eluted from the selected gel slices by crush and soak at 42°C, ethanol precipitated, and resuspended in water. The RNAs were applied to a semipreparative DNAPac PA100 column (Dionex) heated at 65°C (Shields et al 1999) and equilibrated with buffer A (25 mM Tris at pH 7.6 and 2 mM EDTA). They were eluted from the HPLC column using a gradient of buffer B (25 mM Tris at pH 7.6, 2 mM EDTA and 2 M NaCl).…”

Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer

“…The RNA samples for the NMR studies were generated by in vitro transcription using T7 RNA polymerase and synthetic DNA templates (Milligan et al+, 1987;Milligan & Uhlenbeck, 1989)+ The uniformly 13 C/ 15 N-labeled sample of the ⌬-33 RNA was prepared with 13 C/ 15 N-labeled NTPs as described (Batey et al+, 1992;Nikonowicz et al+, 1992)+ Pre-RNAs containing a 7-nt 39-tail sequence (59-GCAGGUC-39) relative to the RNA shown in Figure 1B were transcribed and cleaved with a hammerhead ribozyme added in trans to produce a homogeneous-length RNA as described (Shields et al+, 1999)+ After the cleavage reaction, the product RNA was separated from the pre-RNA by denaturing polyacrylamide gel electrophoresis purification, followed by a DEAE-Sephacel (Phar- macia) anion-exchange chromatography+ The final step in the production of the NMR sample was to dialyze the RNA extensively in the NMR buffer: 20 mM NaH 2 PO 4 , pH ϭ 6+8, 30 mM NaCl, 2 mM MgCl 2 using a Centricon-3 concentrator (Amicon)+ Generally, 10-12 mL of buffer were passed over the RNA to insure complete equilibration of pH and Mg 2ϩ ions+ For the 1:1 RNA-theophylline complex, one equivalent of theophylline (Sigma-reference grade) was added to the sample+ The sample was then lyophilized to dryness and finally resuspended in 350 mL of 90% H 2 O/10% D 2 O or 99+99% D 2 O+ For the NMR studies of the caffeine:RNA complex, 10 equivalents of caffeine were added to the purified RNA sample+ After prolonged storage at 4 8C, samples were heated to 35 8C for 5 min in the NMR tube and then allowed to cool slowly to the experimental temperature+ NMR spectra of the manganese ion titration of RNA-theophylline complex A two-dimensional ( 1 H, 13 C) HSQC spectrum was collected on the uniformly 13 C/ 15 N-labeled RNA/theophylline complex in 20 mM NaH 2 PO 4 , pH ϭ 6+8, 30 mM NaCl, 2 mM MgCl 2 + A Mn 2ϩ titration was performed by adding 5 mL aliquots of a concentrated MnCl 2 solution (in D 2 O), and identical HSQC spectra were collected at total Mn 2ϩ concentrations of 5,10,20,25,30,35, and 40 mM+ The concentration of the RNAtheophylline complex was therefore reduced by ;10% in the 40 mM added Mn 2ϩ spectrum relative to the initial spectrum+ Each spectrum was acquired in ;2 h with 512 complex points for 6,000 Hz in the 1 H dimension (t 2 ), and 190 complex points for 7,500 Hz in the indirect-13 C dimension (t 1 ) and used the States-TPPI method for quadrature detection in t 1 (Marion et al+, 1989)+…”

Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription

“…With the advent of in vitro transcription and the emerging biological significance of short RNA molecules (<40 nt), high-performance liquid chromatography (HPLC) has been applied to purify short RNA transcripts for NMR structural studies (Anderson et al 1996). The addition of the hammerhead ribozyme into the sequence has allowed workers to overcome the length limitation of short RNAs in HPLC purification (Shields et al 1999).…”