Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA

Abstract

A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR.

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)+…”

19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

“…With the exception of imino signals the NMR resonance of the only four different RNA nucleotides resonate in a narrow spectral regime. The limited chemical shift dispersion has-in part-been overcome by multidimensional heteronuclear NMR in combination with uniformly labeling approaches using 13 C, 15 N and 2 H labeled nucleotides in T7 RNA polymerase mediated in vitro transcription (Alvarado et al 2014;Batey et al 1992;Dayie et al 1998;Furtig et al 2008;Nikonowicz et al 1992;Thakur and Dayie 2012) and position specific labeling using chemical synthesis (Kline and Serianni 1990;Neuner et al 2015;Quant et al 1994;Wenter et al 2006).…”

Resolution-optimized NMR measurement of 1DCH, 1DCC and 2DCH residual dipolar couplings in nucleic acid bases

“…bax@nih.gov the introduction of isotopic enrichment procedures for RNA and DNA (Batey et al, 1992;Nikonowicz et al, 1992;Ono et al, 1994;Farmer et al, 1995;Zimmer and Crothers, 1995;Masse et al, 1998) and methods for weakly aligning nucleic acids relative to the magnetic field (Kung et al, 1995;Tjandra and Bax, 1997;Clore et al, 1998;Hansen et al, 1998;Ruckert and Otting, 2000;Sass et al, 2000;Tycko et al, 2000;Chou et al, 2001;Ishii et al, 2001;Meier et al, 2002;Ulmer et al, 2003), residual dipolar coupling (RDC) restraints are now also available. RDCs are particularly useful in nucleic acids, where the number of NOE restraints is typically relatively low, especially those involving long-range contacts.…”