Unambiguous resonance assignments in carbon-13, nitrogen-15-labeled nucleic acids by 3D triple-resonance NMR

Farmer, Bennett T.; Müller, Luciano; Nikonowicz, Edward P.; Pardi, Arthur

doi:10.1021/ja00076a092

Cited by 45 publications

(25 citation statements)

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“…The application of heteronuclear-based NMR experiments to oligonucleotides has provided many dramatic advantages that had previously been realized with isotopical labeling of protein samples. Uniform isotopic labeling of oligonucleotides now allows the unambiguous assignment of resonances using through-bond correlated NMR experiments, like the HCP Marino et al, 1994a;Varani et al, 1995), HCN (Sklenar et al, 1993(Sklenar et al, , 1994(Sklenar et al, , 1996Farmer et al, 1993Farmer et al, , 1994Tate et al, 1993;Simorre et al, 1995Simorre et al, , 1996Marino et al, 1997), and HCP-CCH-TOCSY (Marino et al, 1994b;Wijmenga et al, 1995), and limits reliance on the more ambiguous NOE-based assignment of sequential connectivity. In addition, the extraction of NOE-derived distance restraints is facilitated by the increased resolution obtained by combining NOESY experiments with evolution periods of heteronuclear chemical shifts Varani et al, 1996;Allain and Varani, 1997).…”

Section: Cross-correlated Relaxation For the Measurement Of Angles Bementioning

confidence: 99%

Methods for the Determination of Torsion Angle Restraints in Biomacromolecules

Griesinger

Hennig

Marino

et al. 2002

Modern Techniques in Protein NMR

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Section: Cross-correlated Relaxation For the Measurement Of Angles Bementioning

confidence: 99%

Methods for the Determination of Torsion Angle Restraints in Biomacromolecules

Griesinger

Hennig

Marino

et al. 2002

Modern Techniques in Protein NMR

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“…For example, sugar proton and carbon resonances can be efficiently assigned by application of HCCH-COSY, -RELAY and -TOCSY experiments Nikonowicz and Pardi, 1993); intraresidue base to sugar connectivities can be made by application of HsCsNbCbHb, HsC~Nb, UbNbCs, HbNbC b and HsC~NbHu experiments (Farmer et al, 1993Sklenfi~ et al, 1993a,b;Tate et al, 1994); HCCH-TOCSY experiments can be used to correlate the H2 and H8 resonances in adenine bases Marino et al, 1994a); and sequential resonance assignments can be made by HCP-type and 13C-31p and ~H-3~P heteroTOCSYtype experiments (Kellogg et al, 1992;Heus et al, 1994;Marino et al, 1994bMarino et al, ,1995Wijmenga et al, 1995). Here we present novel 2D and pseudo-3D triple resonance HNCCCH experiments that provide through-bond NH/ NH2 to H6 connectivities for uridine and cytidine base protons in r3C-/~SN-labeled RNAs.…”

mentioning

confidence: 99%

Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cytidine and uridine base protons in RNA

et al. 1995

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A set of triple resonance experiments is presented, providing through-bond H2N/HN to H6 connectivities in uridines and cytidines in 13C-/15N-labeled RNAs. These connectivities provide an important link between the sequential assignment pathways for the exchangeable and nonexchangeable proton resonances in nucleic acids. Both 2D and pseudo-3D HNCCCH experiments were applied to a 30-nucleotide lead-dependent ribozyme, known as the leadzyme. The HN to H6 connectivities for three uridines in the leadzyme were identified from one 2D H(NCCC)H experiment, and the H2N to H6 connectivities were identified for seven of the eight cytidines from the combination of a 2D H(NCCC)H and a pseudo-3D H(NCC)CH experiment.

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“…6A shows the Cl'-Hi' cross peaks in a 2D constant time heteronuclear single quantum correlation (HSQC) (20,21). The proton chemical shifts are correlated to the glycosidic nitrogens in a HCN (22,23) experiment (Fig. 6B), which uses the two consecutive transfers of magnetization from 1H to 13C to '5N and then back.…”

Section: Resultsmentioning

confidence: 99%

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

Zimmer

Crothers

1995

Proc. Natl. Acad. Sci. U.S.A.

123

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Multidimensional heteronuclear NMR experiments can provide a wealth of spectral information in the form of heteronuclear chemical shifts, scalar couplings, and relaxation parameters, which yield structural and dynamic information for macromolecules. In addition, the spectral dispersion provided by the addition of a heteronuclear dimension extends the limit to the size of the macromolecule that can be studied by NMR. 13C and 15N heteronuclei are now routinely incorporated into proteins (1) for use in NMR structure determinations. Recently, the uniform incorporation of these heteronuclei into RNA oligonucleotides has been achieved (2-4). NMR experiments have also been done recently on oligodeoxynucleotides labeled uniformly in the deoxyribose at a single nucleotide (5) and uniformly in a nucleotide (6), but heteronuclear NMR studies of uniformly labeled oligodeoxynucleotides have not been reported because of the technical challenge and the cost of chemical syntheses and polymerization of labeled phosphoramidites.Here we describe an enzymatic synthesis of 13C15N uniformly labeled oligodeoxynucleotides and present some preliminary heteronuclear spectra of a DNA duplex dGGCAAAA CGG&dCCGTTTTGCC (A4.T4), in which either the A4 or T4 strand is uniformly labeled with 13C and 15N. The synthesis procedure involves production of uniformly 13C15N-labeled deoxynucleotides from enzymatic hydrolysis of the DNA of bacteria grown on 99% 13CH30H and >98% 15NH4Cl as sole carbon and nitrogen sources, respectively. The labeled deoxynucleotides are then converted enzymatically to the triphosphates and used in a DNA polymerization reaction that utilizes an oligonucleotide hairpin primer-template containing a ribonucleotide at the 3' terminus. Alkaline hydrolysis of the ribonucleotide linkage between the labeled DNA and the unlabeled primer-template followed by purification yields the labeled DNA. The utility of the heteronuclear labels in DNA is illustrated by heteronuclear NMR spectra of the oligodeoxynucleotide duplex A4-T4.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (2), with the exceptions that cells were grown to stationary phase, DNase I was used, and the nucleotides were put over an anion exchange column prior to separation of the dNMPs from the NMPs. MATERIALS AND METHODS MaterialsGrowth of Methylotrophic Bacteria on 13CH3OH and 15NH4CI. Methylophilus methylotrophus were grown in minimal medium as described (2). After growth to stationary phase and harvesting by centrifugation, a yield of 2 g of wet packed cells per liter was obtained when M. methylotrophus were grown on 13CH30H and 15NH4Cl.Isolation of Nucleic Acids. According to the published procedure (2), 4 g of cells was resuspended in an equal volume of STE (100 mM NaCl/10 mM Tris-HCl/1 mM EDTA, pH 8.0). The cell suspension was added to 100 ml of STE containing 1% SDS and incubated at 37°C for 10 min with sh...

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Unambiguous resonance assignments in carbon-13, nitrogen-15-labeled nucleic acids by 3D triple-resonance NMR

Cited by 45 publications

References 0 publications

Methods for the Determination of Torsion Angle Restraints in Biomacromolecules

Methods for the Determination of Torsion Angle Restraints in Biomacromolecules

Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cytidine and uridine base protons in RNA

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

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