Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography

Hae-Kap Cheong 1 Eunha Hwang 1 Chulhyun Lee 1 Byong-Seok Choi 0 1 Chaejoon Cheong 1 0 Department of Chemistry and National Creative Research Initiative Center, Korea Advanced Institute of Science and Technology , Guseong-dong 373-1, Yuseong-gu. Daejeon 305-701, Korea 1 Magnetic Resonance Team, Korea Basic Science Institute , Eoun-dong 52, Yuseong-gu, Daejeon 305-333, Korea Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in highthroughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 30 end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 30 end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purification method is simpler and faster than the conventional method. - The increasing interest in the structure and function of RNA has created the demand for a high-throughput RNA purification method. For example, X-ray crystallography and NMR spectroscopic studies require milligram quantities of pure RNA in order to obtain useful structural information. The bacteriophage protein T7 RNA polymerase is usually used for in vitro transcription reactions using a synthetic DNA template (1). However, T7 RNA polymerase requires a sequence that starts with G, and some sequences in the first six residues do not give an acceptable yield of RNA transcripts (1,2). RNA purification requires single-nucleotide resolution to separate the transcript of the correct length (N) from aborted (N 1) or add-on (N + 1, N + 2) transcripts, which are usually present in comparable amounts. In most cases, PAGE is used to resolve the correct RNA transcript followed by sizeexclusion liquid chromatography if required. However, the loading capacity of the conventional slab PAGE gel is 60 OD, so the protocols used to obtain milligram quantities of pure RNA for NMR studies require at least four runs on 40 60 0.3 cm3 polyacrylamide gels. In addition, PAGE purification of RNA is tedious, time-consuming and cost ineffective. Immobilized oligonucleotides are widely used in molecular biology, clinical analysis and other biotechnological fields that require the identification of specific DNA sequences (3). Most of these applications are based on hybridization between the immobilized oligonucleotide and complementary sequences in a sample. By constructing columns with a single-stranded polynucleotide covalently attached to the support, the column retains RNA or DNA that can base pair with the attached sequence. This sequence-specific polynucleotide separation technique is called DNA affinity chromatography. The ideal approach for DNA immobilization is covalent binding on a solid surface, via a single-point attachment (4). The type of DNAzyme described here was developed by Santoro and Joyce (5) through in vitro selection. These molecules are DNA oligonucleotides with a small catalytic core that anneals to RNA targets by hybridizing two flanking arms of 713 nt, which each depend on the sequence. On the addition of Mg2+ or other divalent ions, the DNAzyme cleaves the RNA specifically at the designated purine pyrimidine junction. A highly reactive catalytic core has been optimized and kinetically characterized in elegant studies (57). DNAzyme can be targeted to cleave many different RNA sequences efficiently under multiple-turnover conditions (6). In this study, we describe a high-throughput method of preparing RNA samples using DNA affinity chromatography and DNAzyme. To make the purification steps simple and fast, we used sequence-specific affinity column chromatography and a trans-acting DNAzyme. The affinity column chromatography was used to separate abortive transcripts from the correct transcript. The trans-acting DNAzyme eliminated the 30-heterogeneity caused by add-on transcripts. This new method is highly efficient and reliable; 5 mg quantities of an RNA sample can be prepared in three days. MATERIALS AND METHODS Oligonucleotide preparation DNAzyme, complementary DNA oligonucleotides and DNA templates for the T7 RNA polymerase transcription reaction were obtained from Alpha DNA (Canada) and used without further purification. An RNA oligonucleotide, 50-GAGAGUGCUGAUACUGGCCUCUGUAAGAAGCCCUUCAG-30, was synthesized by in vitro transcription with T7 RNA polymerase using unlabeled NTPs and 13C,15N-labeled UTP (1). T7 RNA polymerase and the labeled NTPs were prepared using previously published methods (8,9). The affinity resin was synthesized chemically on 1 g of Oligo Affinity Support (Glen Research). After synthesis, the oligo resin was deprotected in ammonium hydroxide. The concentration of each substrate RNA and DNAzyme was determined by the absorbance at 260 nm. The affinity resin was packed into columns for affinity chromatography. The column was equilibrated with five column volumes of binding buffer (250 mM NaCl, 20 mM sodium phosphate buffer, pH 7.0). The synthesized RNA sample was loaded onto the affinity column and incubated at room temperature for 1 h. The column was washed with five column volumes of binding buffer. The bound RNA was eluted with the elution buffer (20 mM sodium phosphate buffer, pH 7.0). The column was regenerated for the next use by rinsing it several times with elution buffer followed by equilibration with binding buffer. The substrate RNA was combined with DNAzyme in 0.1 reaction buffer (15 mM NaCl, 4 mM TrisHCl, pH 7.5). The solution was heated at 95 C for 34 min to denature the RNA and DNA, and then placed on ice for 5 min. The sample was then incubated at 25 C for 10 min. The cleavage reaction was carried out in 150 mM NaCl, 40 mM TrisHCl (pH 7.5) at 37 C. The reaction was initiated by adding MgCl2 to give a concentration of 60 mM. After the reaction, the DNAzyme DNA was eliminated by digestion with RNasefree DNase I (Promega) followed by size-exclusion chromatography using Superdex 75 (Amersham Biosciences). The products were analyzed by electrophoresis on a 15% polyacrylamide7 M urea denaturing gel. The purified RNA sample was dialyzed extensively against 10 mM sodium phosphate/0.01 mM EDTA (pH 6.8). It was then freeze-dried and dissolved in 0.2 ml of 99.96% D2O. NMR spectra were recorded on a Bruker DMX600 MHz spectrometer, and processed using XWINNMR 3.1 (Bru (...truncated)