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

Abstract

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 high-throughput 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 3' end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 3' 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.

Figure 1

Schematic protocol for preparing high-throughput RNA samples.

Figure 2

The sequence of the oligonucleotides used to prepare an RNA sample.

Figure 3

Purification of RNA with an affinity column and DNAzyme. Lane 1, crude RNA transcription mixture; lane 2, RNA purified using the affinity column; lane 3, the DNAzyme reaction products; lane 4, DNAzyme and product I purified using the affinity column; and lane 5, RNA purified using size-exclusion chromatography. The products were separated by electrophoresis on a 15% polyacrylamide–7 M urea denaturing gel and were detected using toluidine blue.

Figure 4

A section of the constant-time ¹H–¹³C HSQC spectrum of the ¹³C,¹⁵N-UTP-specific labeled RNA recorded at 30°C. The concentration of RNA is 0.8 mM. The sample is in 99.96% D₂O solvent and 10 mM phosphate buffer (pH 6.8). The ¹H dimension is on the horizontal axis, and the ¹³C dimension is on the vertical axis. The C1′s of the uracils resonate at 72–76 ppm, and the C5s at 85–88 ppm. The ¹³C,¹⁵N -UTP-specific labeled U residues in the 23 nt RNA are indicated in boldface type.