Complete 1H Assignments of the Non-Exchangeable Protons of the Non Self-Complementary Heptadeoxyribonucleotide d[(GTCGTCA)·(TGACGAC)] and its Component Strands by High Field NMR

Abstract

The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete ¹H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50°C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the ¹H-NMR experiment was established independently of the NMR evidence by ³²P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete ¹H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.     

1H and 31P-NMR assignments of the non-exchangeable protons of the consensus acceptor exon:intron junction d(CpTpApCpApGpGpT)

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.     

1H and 31P-NMR Assignments of the Non-exchangeable Protons of the Consensus Acceptor Exon: Intron Junction d(CpTpApCpApGpGpT)

Abstract

The consensus acceptor exon: intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20°C as evidenced by temperature variable ¹H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique ¹H-¹H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of ³¹P-¹H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon: intron junction adopts a random coil conformation in solution under the experimental conditions employed.     

Synthesis, complete 1H assignments and conformations of the self-complementary hexadeoxyribonucleotide [d(CpGpApTpCpG)]2 and its fragments by high field NMR.

The two deoxyribonucleotides [d(CpGpApTpCpG)]2 and [d(CpGpCpG)]2 were synthesized by the phosphotriester method. Their duplex form under the conditions of the 1H-nmr experiments was proven by end 32P labeling with T4 polynucleotide kinase followed by butt end joining employing the absolute specificity of T4 ligase for double stranded DNA and analysis using gel electrophoresis and autoradiography. Complete nmr assignment of the 1H chemical shifts and coupling constants was achieved. The assignments were secured using sequential decoupling, NOE difference measurements, and two-dimensional COSY and SECSY experiments. Spectrum simulation confirmed the experimental values of chemical shifts and coupling constants. The techniques for the assignment outlined together with 31P and 2-D heteronuclear shift correlation permit an approach to a systematic analysis of more complex single-strand and duplex oligodeoxyribonucleotides.     

Two-dimensional 1H-NMR investigation of ribonuclease T1. Resonance assignments, secondary and low-resolution tertiary structures of ribonuclease T1

Ribonuclease T1 was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequence-specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and C beta protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects.     

1H-NMR assignments of the non-exchangeable protons of the consensus donor exon:intron junctioń d(CpApGpGpTpApApGpT)

The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20 degrees C was confirmed by temperature variable 1H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T1 experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.     

1H-NMR Assignments of the Non-exchangeable Protons of the Consensus Donor Exon:Intron Junction d(CpApGpGpTpApApGpT)

Abstract

The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20°C was confirmed by temperature variable ¹H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T₁ experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.     

Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC).

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.     

Structure and conformation of the duplex consensus acceptor exon:intron junction d[(CpTpApCpApGpGpT). (ApCpCpTpGpTpApG)] deduced from high-field 1H-NMR of non-exchangeable and imino protons

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.     

Core region of Citrobacter lipopolysaccharide from strain PCM 1487. Structure elucidation by two-dimensional 1H-NMR spectroscopy at 500 MHz and methylation analysis/mass spectrometry

The core structure of Citrobacter PCM 1487 lipopolysaccharide has been established using methylation analysis/mass spectrometry, chemical degradations and one- and two-dimensional 1H-NMR spectroscopy at 500 MHz. 1H-NMR assignments are given for all sugar components of the core oligosaccharide. In the formula shown below, the alternative locations of branch terminal heptose (LDHep) and diphosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (Formula: see text). The sample of the core oligosaccharide showed some microheterogeneity due to a slightly incomplete substitution by terminal N-acetylgalactosamine and a partial splitting of diphosphorylethanolamine residues.     

Sequence dependent conformation and local geometry of the conserved branch site sequence element d(TpApCpTpApApC), essential for yeast mRNA splicing, deduced from high resolution 1H-NMR

The conserved sequence element and branch site splice signal d(TpApCpTpApApC) has been synthesized by a solid phase procedure. All the non-exchangeable protons have been assigned using a combination of one-dimensional and two-dimensional 1H-NMR analytical procedures. On the basis of the low NOE intensities in the 1D-NOE and NOESY experiments, the heptamer exists in solution as a random coil. The deoxyribose rings towards the 5' terminus exist predominantly in the S form (2'-endo-3'-exo) while residues on or adjacent to the 2' branch site in the eventual lariat structure [A(6) of TACTAAC] show more N-character (3'endo-2'-exo). In addition unique propeller twisting at contiguous AT base pairs in the consensus 5'-splice site occurs in the region in which there is partial complementarity with the branch splice signal TACTAAC. These subtle structural features, if carried over to the corresponding RNA, may have significance either as a recognition signals or for stereochemical reasons in the formation of the lariat intermediate in the maturation process of mRNA.     

Study of the interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in aqueous solution by uni- and two-dimensional 1H-spectroscopy]

Complex formation between acridine dye proflavine and self-complementary deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water-salt solution was studied by the method of one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Two-dimensional homonuclear 1H-NMR spectroscopy (2D-COSY and 2D-NOESY) was used for complete assignments of proton signals of molecules in solution and for qualitative analysis of the nature of interactions between proflavine and tetranucleotide. Concentration dependences of proton chemical shifts of the molecules were measured at 293 K. Equilibrium reaction constants and limiting chemical shifts of dye protons in the complexes were determined using suggested schemes of complex formation. Based on the obtained data possible types of complexes were considered. Analysis of relative content of different types of complexes was made and special features of dynamic equilibrium were revealed as a function of correlation of dye and tetranucleotide concentrations. The most favourable structure of 1:2 complex of dye with tetranucleotide was constructed using the calculated values of induced chemical shifts of proflavine protons and 2D-NOESY spectra.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transferrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The 1H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional 1H-1H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503-506).     

Sequence-specific assignment of 1H-NMR resonance and determination of the secondary structure of Jingzhaotoxin-I

Jingzhaotoxin-I （JZTX-I） purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3.The structure of this toxin in aqueous solution was investigated using 2-D ^1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the ^1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in n20 and D20. All the backbone protons except for Gin4 and more than 95% of the side-chain protons were identified by dαN,dαδ, dβN and dNN connectivities in the NOESY spectrum. These studies provide a basis for the furtherdeter mination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel β-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I （HWTX-I） and hainantoxin-IV （HNTX-IV）, whose structures in solution havepre viously been reported.     

1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.     

1H-NMR assignments and local environments of aromatic residues in bovine, human and guinea pig variants of alpha-lactalbumin.

1H-NMR assignments have been defined for the aromatic-ring protons of the bovine, guinea pig and human variants of alpha-lactalbumin. Spin-system networks were identified by means of double-quantum-filtered two-dimensional J-correlated spectroscopy and two-dimensional relayed coherence spectroscopy data. Analysis of two-dimensional nuclear-Overhauser-enhancement spectroscopy data of the proteins indicated that in each case two clusters of aromatic residues exist. The two clusters are also evident in the crystal structure of the human protein, and this evidence, in conjunction with sequence differences between the three proteins, permitted sequence-specific assignments to be made for the majority of aromatic residues. Remaining ambiguities in the assignments could be resolved by analysis of photochemically induced dynamic nuclear polarization (PCIDNP) effects. Comparison of the PCIDNP spectra of the three proteins indicated the presence of only minor differences in the surface exposure of conserved aromatic residues. Taken together, these results indicate that the environments of the conserved aromatic residues in bovine, guinea pig and human alpha-lactalbumin in solution are very similar to each other, and that the solution and the crystal forms of at least the human protein are similar.     

Structure and Conformation of the Duplex Consensus Acceptor Exon: Intron Junction d [(CpTpApCpApGpGpT) (ApCpCpTpGpTpApG)] deduced from High-Field 1H-NMR of Non-Exchangeable and Imino Protons

Abstract

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20°C as evidenced by temperature variable ¹H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and ¹H-¹H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D₂O:H₂O mixtures. Assignment of the nonexchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5′ regions exist predominantly in the S form (2′endo—3′exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2′exo—3′endo). The contiguous bases A₅-G₆ (adjacent to the junction) and A₁₅-G₁₆ are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transffrrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The ¹H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional ¹H-¹H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503–506).