Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N

Summary It is demonstrated that sequential resonance assignment of the backbone ¹H and ¹⁵N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate ¹H with the intraresidue ¹⁵N and with the ¹⁵N resonance of the next residue. The experiments are preferably conducted in D₂O, where very high resolution in the ¹⁵N dimension can be achieved by using ²H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in ¹³C and ¹⁵N. Complete backbone and ¹³C/¹H resonance assignments are presented.     

Novel projected 4D triple resonance experiments for polypeptide backbone chemical shift assignment

Here we present a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment of polypeptide backbone chemical shifts in ¹³C/¹⁵N doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments, the ¹³C and ¹H chemical shifts evolve in a common dimension and are simultaneously detected in quadrature. These experiments are particularly useful for the assignment of glycine-rich polypeptide segments. Appropriate setting of the ¹H radiofrequency carrier allows one to place cross peaks correlating either backbone ¹⁵N/¹H^N/¹³C or ¹⁵N/¹H^N/¹H chemical shifts in separate spectral regions. Hence, peak overlap is not increased when compared with the conventional 3D HNNCA and HNN(CA)HA. 3D HNN[CAHA] and 3D HNN(CO)[CAHA] are complemented by 3D reduced-dimensionality (RD) HNN COCA and HNN CACO, where ¹³C and ¹³C chemical shifts evolve in a common dimension. The ¹³C shift is detected in quadrature, which yields peak pairs encoding the ¹³C chemical shift in an in-phase splitting. This suite of four experiments promises to be of value for automated high-throughput NMR structure determination in structural genomics, where the requirement to independently sample many indirect dimensions in a large number of NMR experiments may prevent one from accurately adjusting NMR measurement times to spectrometer sensitivity.     

Sensitivity enhancement in (HCA)CONH experiments

A novel sensitivity-enhancement technique is proposed for experiments which correlate protein backbone resonances and start with magnetization from ¹³C-¹H groups. The technique is based on replenishing magnetization lost by dipole-CSA cross-correlated relaxation of the ¹³C spin with ¹³C steady state magnetization. The principle is demonstrated for the (HCA)CONH experiment, resulting in 1.6-fold sensitivity enhancement compared to the HN(CA)CO experiment. Furthermore, other versions of the (HCA)CONH experiment were evaluated, including a novel experiment with spin-locking of transverse ¹³C-¹H two-spin coherence, and a cross-correlation compensated (CA)CONH experiment which starts from ¹³C rather than ¹H magnetization.     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

A simple urea leaf-feeding method for the production of 13C and 15N labelled plant material

The use of ¹³C isotope tracer techniques in terrestrial ecology has been restricted by the technical requirements and high costs associated with the production of ¹³C enriched plant material by ¹³CO₂ release in labelling chambers. We describe a novel, simple and relatively inexpensive method for the small-scale production of ¹³C and ¹⁵N labelled plant material. The method is based on foliar feeding of plants with a urea solution (97 atom% ¹³C, 2 atom% ¹⁵N) by daily misting. Maize was grown in a greenhouse in a compost–soil mixture and enclosed in clear polythene bags between urea applications. Final enrichment in 27 d old maize shoots was 211 ¹³C (1.34 atom% ¹³C) and 434 ¹⁵N (0.52 atom% ¹⁵N). Enrichments of hot-water extractable fractions (289 ¹³C, 469 ¹⁵N) were only slightly higher than those observed in plant bulk material, which suggests that daily urea applications ensured fairly uniform labelling of different biochemical fractions and plant tissues. Recovery of applied excess ¹³C and ¹⁵N in plant shoots was 22% and 42%, respectively. Roots were less enriched (21 ¹³C and 277 ¹⁵N), but no attempts were made to recover roots quantitatively.     

Tracing mangrove carbon in suspended matter and aquatic fauna of the Gautami–Godavari Delta,Bay of Bengal (India)

Stable carbon isotopic composition and C/N ratio were used to trace the input of carbon associated with mangrove litter into the estuary of the Godavari–Gautami delta system and Kakinada bay (Andhra Pradesh, India). Suspended organic matter in the mangrove channels was more depleted in ¹³C (average ¹³C = –24.5) than in Kakinada bay which showed ¹³C values for suspended matter (average ¹³C = –22.7) closer to those expected for marine phytoplankton. Suspended organic matter from mangrove channels was enriched in nitrogen (average C/N atom ratio 12.7) and ¹³C (average ¹³C = –24.5) relative to mangrove leaf litter, which had a C/N ratio of 75 and a ¹³C value of –28. Lowest C/N ratios for suspended matter were observed during southwest monsoon when rainfall was highest. Although in general, mangrove litter fall was also lower during this period, no clear correlation was observed between litter fall and C/N ratio of suspended matter. In general, the composition of suspended matter pointed towards phytoplankton as a major component. Isotopic composition of zooplankton suggested selective feeding on ¹³C-enriched, marine phytoplankton in open Kakinada bay and on ¹³C-depleted organic matter, such as estuarine phytoplankton and mangrove litter, in the mangrove channels. From the ¹³C signature, it appeared that mangrove carbon was present to some extent in zooplankton and macrofauna from the mangrove mudflats and channels, but the signal rapidly decreased in Kakinada bay. Nitrogen isotopic composition of zooplankton and macrofauna indicated a progressive enrichment of ¹⁵N away from the mangrove forest towards the northern part of Kakinada bay, in approach of Kakinada city. This is thought to reflect input of anthropogenic nitrogen enriched in ¹⁵N and subsequent uptake of this enriched nitrogen into the aquatic food chain.     

Backbone resonance assignment in large protonated proteins using a combination of new 3D TROSY-HN(CA)HA, 4D TROSY-HACANH and 13C-detected HACACO experiments

A new method for backbone resonance assignment suitable for large proteins with the natural ¹H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, ¹³C-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the ¹³C antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly ¹⁵N,¹³C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins.     

A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on ¹⁵N, natural abundance ¹³C samples. The experiment exploits the fact that transfers of magnetization from ¹⁵N to ¹³CO and from ¹⁵N to ¹³C (and back) proceed independently for samples that are not enriched in ¹³C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM ¹⁵N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz.     

Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.Human infections with the swine origin influenza virus A (H1N1) were first detected in April 2009 and spread across the globe, resulting in WHO declaring a pandemic on 12 June 2009 for the first time in the past 41 years. More than 296,471 people have had confirmed infections with this novel H1N1 virus, and there have been at least 3,486 deaths as of September 18, 2009. In the last century, an influenza H1N1 virus caused the devastating 1918-1919 pandemic; this pandemic was characterized by a mild outbreak in the spring of 1918, followed by a lethal wave globally in the fall of that year which killed as many as 50 million people worldwide (20, 29). The 2009 H1N1 viruses circulating globally since April 2009 have not caused a significant rise in mortality related to influenza. Nucleotide sequence analysis suggested that E627 in PB2, a deletion of the PDZ ligand domain in NS1, and the lack of the PB1-F2 open reading frame in the 2009 H1N1 viruses may contribute to the relatively mild virulence (20, 26, 27). Recent animal studies have shown that the 2009 H1N1 influenza viruses did not replicate in tissues beyond the respiratory tract and did not cause significant mortality in the ferret model; however, the 2009 H1N1 viruses are capable of infecting deep in the lung tissues and caused more significant lesions in the lung tissues of animals, including nonhuman primates, than typical seasonal strains (13, 17, 19). Children and young adults are particularly susceptible to the 2009 H1N1 virus infection because they have no or low immunity to the novel 2009 H1N1 strains (11, 13). The widespread and rapid distribution of the 2009 H1N1 viruses in humans raises a concern about the evolution of more virulent strains during passage in the population. One fear is that mutant forms of the 2009 H1N1 viruses may exhibit significantly increased virulence (2, 19). Therefore, there is an urgent need to develop an effective vaccine to control the influenza pandemic caused by the swine origin H1N1 viruses.Live attenuated influenza vaccine (LAIV) has been licensed in the United States annually since 2003. The seasonal vaccine protects against influenza illness and elicits both systemic and mucosal immune responses, including serum hemagglutination inhibition (HAI) antibodies that react to antigenically drifted strains (3, 4). A critical attribute of an effective pandemic vaccine is its capability to elicit an immune response in immunonaive individuals; LAIV has been shown to offer protection following a single dose in young children. However, two doses of vaccines are recommended for children younger than 9 years of age who have never been immunized with influenza vaccines. In order to produce LAIV to protect against the newly emerged swine origin H1N1 influenza virus, we have produced several 6:2 reassortant candidate vaccine strains that express the hemagglutinin (HA) and neuraminidase (NA) gene segments from influenza virus A/California/4/09 (A/CA/4/09) (H1N1) or A/CA/7/09 (H1N1), as well as the six internal protein gene segments (PB1, PB2, PA, NP, M, and NS) from cold-adapted A/Ann Arbor/6/60 (H2N2) (AA60) virus, which is the master donor virus for all influenza virus A strains in trivalent seasonal LAIV. Initial evaluation of these candidate vaccine strains indicated that they did not replicate as efficiently as seasonal H1N1 influenza vaccine strains in embryonated chicken eggs. In this report, we describe directed modifications of the HA gene segment that improved vaccine yields in eggs, resulting in a number of vaccine candidates that are available for human use.     

Effects of competition and N and P supply on carbon isotope discrimination and <Superscript>15</Superscript>N-natural abundance in four grassland species

The effect of interspecific competition and element additions (N and P) on four grassland species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale) grown under field conditions was studied. Two grasses (L. perenne, F. valida) grown in monoculture (absence of competition) showed lower carbon isotope discrimination (¹³C) and enriched ¹⁵N values. Nitrogen addition (as urea) had inconsistent effects on species ¹³C while caused enrichment of ¹⁵N of P. pratensis and F. valida but strong depletion of ¹⁵N of T. officinale. Phosphorous had no significant effect on ¹³C but depleted ¹⁵N of all species.     

Novel 2D and 3D multiple-quantum bi-directional HCNCH experiments for the correlation of ribose and base protons/carbons in 13C/15N labeled RNA

Multiple-quantum 2D and 3D bi-directional HCNCH experiments are presented for the correlation of base and ribose protons/carbons in ¹³C/¹⁵N labeled HIV-1 TAR RNA. In both 2D and 3D experiments, the magnetization of H1 is transferred to H6/H8 and H1 through H1-C1-N1/9-C6/8-H6/8 and H1-C1-N1/9-C1-H1 pathways, and the magnetization of H6/8 is transferred to H1 and H6/8 through H6/8-C6/8-N1/9-C1-H1 and H6/8-C6/8-N1/9-C6/8-H6/8 pathways. Chemical shifts of four different nuclei (H1, C1, C6/8 and H6/8) are sampled in the 2D experiment. The correlation of base and ribose protons/carbons is established by the rectangular arrangement of crossover and out-and-back peaks in the proton/carbon correlated spectrum. The rectangular connections can be further resolved using the nitrogen dimension in a ¹H/¹³C/¹⁵N 3D experiment. Furthermore, by taking advantage of the well separated chemical shifts of N1 (pyrimidine) and N9 (purine), the 2D spectrum can be simplified into two sub-spectra based on their base type. Both experiments were tested on a ¹³C/¹⁵N labeled 27-mer HIV-1 TAR RNA containing a UUCG hairpin loop.     

Regioselective addition of bifunctional oximehydroxamic acid by the hydroxamic group to Pt(IV)-coordinated nitriles

The metal-mediated coupling between the nitriles RCN in the platinum(IV) complexes trans-[PtCl₄(RCN)₂] (RMe, Et, CH₂Ph, Ph), cis/trans-[PtCl₄(MeCN)(Me₂SO)] and the newly synthesized bifunctional oximehydroxamic acid, viz. N,2-dihydroxy-5-(1-hydroxyiminoethyl)benzamide, proceeds smoothly in CH₂Cl₂ at 40-45 °C to accomplish the new metallaligands HNC(R)ONHC(O)C₆H₃(2-OH)(5-C(Me)NOH) with pendant oxime functionalities due to the regioselective addition of the reagent via its hydroxamic groups. The obtained iminoligands exist in hydroxamic/hydroximic tautomeric equilibrium in solution. The structures of the isolated compounds are based on elemental analyses (C, H, N), IR, 1D ¹H, ¹³C{¹H}, and 2D NMR correlation experiments, i.e. ¹H,¹³C-COSY, ¹H,¹³C long range COSY, ¹H,¹⁵N-COSY, and ¹H,¹⁵N long range COSY.     

Unambiguous through-bond sugar-to-base correlations for purines in 13C, 15N-labeled nucleic acids: The HsCsNb,HsCs(N)bCb,and HbNbCb experiments

Summary A set of three 3D (¹H, ¹³C, ¹⁵N) triple-resonance correlation experiments has been designed to provide H1-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the H_sC_sN_b, H_sC_s(N)_bC_b, and H_bN_bC_b experiments correlate the H1 sugar proton to the H8 proton of the attached base by means of the {H1, C1, N9, C8, H8} heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1-H8 intraresidue correlations, provided that no two purines have both the same H1 and C1 chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1-H8 intraresidue sugar-to-base correlations for all five guanosines in the [¹³C, ¹⁵N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)₂.     

Intraresidue 1H-15N-13C′ and 1Hα-13Cα-13C′ dipole-CSA relaxation interference as a source of constraints for structural refinement of metal-binding sites in zinc-finger proteins

¹H(i)-¹⁵N(i)-¹³C(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the ¹³C,¹⁵N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane torsion angles and taken from the solution structure. ¹H(i)-¹⁵N(i)-¹³C(i) as well as ¹³C(i)-¹H(i)-¹³C(i) dipole-CSA cross-correlation rates were subsequently used to improve the geometry of the metal binding site. The optimized dihedral angles of the two zinc-binding sites in qCRP2(LIM2) are in better agreement with values obtained from crystal structures of other zinc-finger proteins and thus establish the utility of this approach to improve the metal-binding site geometry of zinc-finger proteins studied by NMR spectroscopy in solution.     

A 4D TROSY-based pulse scheme for correlating 1HNi,15Ni,13Cαi,13C′i−1 chemical shifts in high molecular weight, 15N,13C, 2H labeled proteins

A 4D TROSY-based triple resonance experiment, 4D-HNCO_i–1CA_i, is presented which correlates intra-residue ¹HN, ¹⁵N, ¹³ C chemical shifts with the carbonyl (¹³C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (¹HN,¹⁵N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO_i–1CA_i often allows resolution of the ambiguity by linking the ¹³C and ¹³C spins surrounding the (¹HN,¹⁵N) pair. The experiment is demonstrated on a sample of ¹⁵N, ¹³C, ² H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns.     

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA

Deuterium isotope effects and fractionation factors of N1...H3–N3 hydrogen bonded Watson–Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of ¹³C2 and ¹³C4, ²¹³C2 and ²¹³C4, and equilibrium deuterium/protium fractionation factors of H3, , were measured and seen to correlate with the chemical shift of the corresponding imino proton, _H3. Downfield-shifted imino protons associated with larger values of ²¹³C2 and ²¹³C4 and smaller values, which together suggested that the effective H3–N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that ²¹³C2, ²¹³C4 and values can be useful gauges of hydrogen bond strength of A:T base pairs.     

Improved 3D gd-HCACO and gd-(H)CACO-TOCSY experiments for isotopically enriched proteins dissolved in H2O

Summary Pulsed field gradients were incorporated into the HCACO experiment for acquiring spectra on isotopically enriched protein samples dissolved in H₂O. Excellent water suppression and spectral quality were achieved using the modified pulse sequence (gd-HCACO), as demonstrated for a ¹³C-/¹⁵N-labeled sample of the SH2 domain from the hematopoietic cellular kinase dissolved in 90% H₂O/10% D₂O. Strong correlations for all residues were observed in the gd-HCACO spectrum, even for residues having -protons resonating exactly at the H₂O frequency. The HCACO-TOCSY experiment was modified to correlate intraresidue ¹³C (rather than ¹H), carbonyl (¹³C), and aliphatic side-chain protons [(H)CACO-TOCSY]. Pulsed field gradients were also incorporated into the (H)CACO-TOCSY experiment for water suppression.     

Enantioselective syntheses of isotopically labelledα-amino acids Preparation of specifically13C-labelled L-lysines

Summary [2-¹³C]-L-lysine, [3,4-¹³C₂]-L-lysine and [5,6-¹³C₂]-L-lysine are prepared from simple, commercially available, highly enriched starting materials as [2-¹³C]-glycine, ethyl [1,2-¹³C₂]-bromo acetate, and [1,2-¹³C₂]-acetonitrile. The introduction of the chiral center is based on a general method starting from the bis-lactim ether of cyclo-(D-Val-Gly). The synthesis of (2R)-[5-¹³C]-3,6-diethoxy-2,5-dihydro-2-isopropylpyrazine is described. The availability of our method for the preparation of specifically enriched bis-lactim ethers allows the synthesis of a great variety of site specific isotopically labelled (L- and D-)-amino acids. Moreover, intermediate 4-[(2R,5S)-3,6-diethoxy-2,5-dihydro-2-isopropyl-5-pyrazinyl]butyronitrile is a valuable precursor in the synthesis of L--aminoadipic acid. The synthetic scheme in this publication makes both L-lysine and L--aminoadipic acid¹³C- or¹⁵N-labelled at any position, easily available. The isotopomers of lysine are obtained on a preparative scale in good yields, with 99%¹³C and high enantiomeric purity (>97% e.e.). Three isotopomers are characterized using various spectroscopic techniques,e.g.,¹H NMR,¹³C NMR and Mass spectrometry.     

Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments

Summary The advent of methods for preparing ¹⁵N- and ¹³C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the ¹³C label. It may therefore sometimes be desirable to prepare a relatively inexpensive ¹⁵N-labeled sample only. Here we show that the two-bond ¹H-¹⁵N HSQC experiment can be used on ¹⁵N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected ¹H-¹⁵N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.