Assignment of NH resonances in nucleic acids using natural abundance ¹⁵N‐¹H correlation spectroscopy with spin‐echo and gradient pulses

Abstract

It is well known that 2D 15N‐1H correlation spectra can resolve overlapped imino proton resonances in the downfleld NMR spectra of nucleic acids according to their 15N chemical shifts, and that these resonances can be assigned by base type on that basis, independent of conformation. This information can be extremely important in determining the solution structure of a nucleic acid by NMR, but previously could only be obtained using 15N‐labeled, or very concentrated samples. Here we report the design of a gradient‐enhanced, jump‐return spin echo version of an 15N‐1H HMQC experiment (GE‐JRSE HMQC) that is sensitive enough to work on unlabeled nucleic acid samples at normal NMR concentrations. This experiment has led to the assignment of imino proton resonances with non‐Watson‐Crick chemical shifts in the spectrum of a 29 residue oligoribonucleotide that models the sarcin/ricin loop from 28S rRNA.

NMR and biochemical characterization of recombinant human tRNA3 Lys expressed in Escherichia coli: Identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription

“…The TOCSY (Griesinger et al+, 1988) spectrum in D 2 O was acquired at 600 MHz and 35 8C with 2,048 data points in the t2 dimension and 512 t1 increments+ The spectral width was 6,000 Hz in both dimensions+ A homonuclear proton spin lock with a MLEV-17 pulse sequence (Bax & Davis, 1985) was applied during a mixing time of 40 ms+ A 1 H-15 N HMQC experiment (Szewczak et al+, 1993) was carried out at 800 MHz and 15 8C with 128 t1 increments and 1,024 data points per increment+ The spectral widths were 6,000 Hz in the 1 H dimension and 2,000 Hz in the 15 N dimension+ The solvent signal suppression was realized as in the NOESY experiment+ An HNCO experiment (Kay et al+, 1990) was carried out at 600 MHz and 15 8C in a two-dimensional version with 1 H and 13 C dimensions+ We acquired 1,024 data points for 256 increments in the 13 C dimension and 384 scans per increment+ The high rate of exchange of imino protons with water makes this experiment less sensitive for nucleic acids than for proteins+ To avoid saturation of the exchangeable protons of tRNA, water suppression was achieved using flip-back pulses (Grzesiek & Bax, 1993)+ The spectral widths were 6,000 Hz in the 1 H dimension and 7,545 Hz in the 13 C dimension+ The 1 H carrier frequency was set in the middle of the region of the exchangeable proton resonances+ Protons were decoupled from 15 N during acquisition using the GARP sequence (Shaka et al+, 1985)+…”

Three‐Dimensional Solution Structure of Human Angiogenin Determined by 1H,15N‐NMR Spectroscopy — Characterisation of Histidine Protonation States and Pka Values

“…Exchange of amide protons with the solvent was monitored by recording at 303 K a series of IH-"N HMQC spectra (Szewczak et al, 1993) over a period of 90 h. A sample of lSN-labelled angiogenin was freezedried and resuspended in the same volume of 100% *H,O. The first HMQC spectrum was started within 30 min after dissolving the protein in the deuterated solvent.…”

Determining RNA solution structure by segmental isotopic labeling and NMR: application to Caenorhabditis elegans spliced leader RNA 1.

“…The 2D NOESY spectra in H20 were obtained in the pure absorption mode (21) using a standard pulse sequence (22), except that the last 900 pulse was replaced with a gradient-enhanced jump-return spin-echo sequence for water suppression (19,20). The 2D {15N}1H heteronuclear multiple quantum correlation (HMQC) was collected for imino protons using a gradient-enhanced jump-return spinecho pulse sequence (23).…”