NMR studies of tris-intercalation: solution structure and interaction of d(CTTCGCGCGAAG) with an acridine trimer

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of 1H and 31P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H5', H5"), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O1'endo range, those of the internal guanosines being closer to C2'endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the 3T4C5G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3'-5')G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

Nuclear magnetic resonance investigation of lysine–oligopeptides and a complex with d(pA)3pGpC(pT)3

We report proton magnetic resonance studies of a series of lysine oligopeptides in H₂O solution. At pH 5 the protonated ε-amino groups are seen as broad resonances; the peptide NH proton resonances are split by spin–spin coupling with the C^α-H proton, and appear at positions which depend on position in the chain and on chain length. Assignments were made by the europium shift method, and we observed the expected effect of catalysis by the terminal —NH₃⁺ of exchange of the adjacent peptide NH. Coupling constants and the temperature coefficient of chemical shift values were consistent with a non-hydrogen-bonded structure for the oligolysines. The rate and mechanism of NH hydrogen exchange were investigated by line-broadening measurements of the peptide protons as a function of pH. Exchange was found to be OH^? catalyzed, with large differences in the rate depending on position in the chain. Preliminary studies of the complex between double-helical d(pA)₃pGpC(pT)₃ and tetra(L -lysine) were performed using ¹H- and ³¹P-nmr techniques. Pmr spectra of the complex at pH values ranging from 3.98 to 8.15 showed very complicated patterns. Downfield shifts and reduction in exchange rates were observed for several tetra(L -lysine) protons. ³¹P-nmr spectra of the complex reveal an upfield shift of 1 ppm for 3′-5′ phosphate diester resonances on complexation. ³¹P T₁ relaxation times change little on complex formation at low temperature but are altered at higher temperature.     

Ab-inito Quantum Mechanical Calculations of NMR Chemical Shifts in Nucleic Acids Constituents II Conformational Dependence of the lH and 13C Chemical Shifts in the Ribose

Abstract

The magnetic shielding constant of the different ¹³C and ¹³H nuclei of a deoxyribose are calculated for the C2′ endo and C3′ endo puckerings of the furanose ring as a function of the conformation about the C4′C5′ bond. For the carbons the calculated variations are of several ppm, the C3′ endo puckering corresponding in most cases to a larger shielding than the C2′ endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose ?3′ and 5′ phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides.

The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

Base-pair exchange kinetics of the imino and amino protons of the 3′-phenazinium tethered DNA-RNA duplex,r(5′GAUUGAA3′):d(5′TCAATC3′-Pzn), and their comparison with those of B-DNA duplex

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [^5′r(G¹A²U³U⁴G⁵A⁶A⁷)^3′]:^5′p[d(T⁸C⁹A¹⁰A¹¹T¹²C¹³)]^3′-Pzn] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C¹G²T³A⁴C⁵G⁶)₂ (II), d[p(^5′T¹G²T³T⁴T⁵G⁶ G⁷C⁸)^3′]:d[p(^5′C⁹C¹⁰A¹¹A¹²A¹³C¹⁴A¹⁵)^3′] (III), d(C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶)₂ (IV) and d(C¹G²C³G⁴C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶C¹⁷G¹⁸C¹⁹G²⁰)₂ (V). This comparative study shows that the life-times τ_o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of ∼ 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the τ_o of the larger duplexes (IV) and (V): the τ_o for the base pair of T¹¹ and T¹² residues in the 20-mer (duplex V) are 2.9 ± 2.3 ms and 23.2 ± 8.9 ms, respectively, while the corresponding τ_o in the 12-mer (duplex IV) are 2.8 ± 2.2 ms and 17.4 ± 5.4 ms. It has also been shown that the total energy of activation (E_a) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the E_a of the short B-DNA duplex (E_a ≈ 28–47 kcal/mol).     

The Effect of Crystal Packing on Oligonucleotide Double Helix Structure

Abstract

Complexes formed between Actinomycin D (ActD) and the tetranucleotides d(AGCT)2 and d(CGCG)₂ were studied in detail by one and two-dimensional ¹H and ³¹P NMR. The ³¹P two dimensional chemical exchange experiment, at room temperature on saturated complexes (1:1), showed unambiguously that the asymmetrical phenoxazone ring binds to the unique GC site under the two possible orientations in the d(AGCT)₂ tetranucleotide but adopts a single orientation in the d(CGCG)₂ tetranucleotide. For the d(CGCG)₂: Act D saturated complex, complete assignments of all protons and phosphorus signals of the two-nucleotide strands, as well as of the two cyclic pentapeptide chains has allowed us to study in details the conformational features of the complex from NOE and coupling constants analysis. The tetranucleotide remains in a right-handed duplex, but the sugar puckers are modified for residues at the intercalation site. A uniform C_2′ endo pucker is observed for residues on the strand facing the quinoid side of the phenoxazone ring while a C_2′ endo-C_3′ endo equilibrium about 60% of C_2′ endo is proposed for the two residues on the strand facing the benzenoid side of the phenoxazone ring. In contrast to previous studies on ActD-DNA interactions, we have been able to measure the ³J phosphorus-proton coupling constants at the intercalation site but also adjacent to it, showing that ³¹P chemical shifts are not simply related to the backbone conformation. Molecular mechanics calculations, using empirical distances deduced from NOE effects as restrained distances during minimizations, led to a model differing mainly from those previously published by orientation of the N methyl groups of both N-Methyl-Valines.     

Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field

For base-paired nucleic acids, variations in ¹ J _NH and the imino ¹H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the ¹ J _NH coupling for the imino proton then can be approximated by ¹ J _NH = (1.21Hz/ppm)δ_H − 103.5 ± 0.6 Hz, where δ_H represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (¹ J _NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA^Val and the alignment tensor determined from magnetic-field alignment of tRNA^Val agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

High resolution nuclear magnetic resonance spectroscopy of glycosphingolipids. I: 360 MHz 1H and 90.5 MHz 13C NMR analysis of galactosylceramides

The high resolution ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of galactosylceramides containing n-fatty acids and α-hydroxy fatty acids were recorded in dimethylsulfoxide solution with and without addition of D₂O. From the coupling constants of the sugar ring protons, a ⁴C₁ conformation can be deduced. In contrast to the conformation in aqueous solution, the C⁶ hydroxymethylene group is freely rotating around the C⁶C⁵ bond. In the ceramide residue all signals produced by protons linked to carbons bearing electronegative substituents could be attributed. The large difference in coupling constants of the methylene protons of C^1′ to the C^2′ methine proton of the sphingosine indicates a restricted rotation around the C^1′C^2′ bond. The assignments of the hydroxy and amino protons follow from the decoupling of the corresponding methine protons.     

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.     

Long-range imino proton-13C J-couplings and the through-bond correlation of imino and non-exchangeable protons in unlabeled DNA

Thanks to rather large (5–9 Hz) long-range imino proton-¹³C J-couplings, heteronuclear correlation experiments in H₂O provide unambiguous assignment of imino protons by intranucleotide through-bond connectivities to guanosine H8 and thymidine CH₃ protons, or sequence-specific assignment of non-exchangeable protons when the imino protons are identified independently. This method is demonstrated in the Dickerson dodecamer [d(CGCGAATTCGCG)]₂ and in a human telomeric fragment of 22 nucleotides.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

d-CpCpGpG and d-GpGpCpC self-complementary duplexes: Nmr studies of the helix-coil transition

We have monitored the helix-coil transition of the self-complementary d-CpCpGpG and d-GpGpCpC sequences (20mM strand concentration) at the base pairs, sugar rings, and backbone phosphates by 360-MHz proton and 145.7-MHz phosphorus nmr spectroscopy in 0.1M phosphate solution between 5 and 95°C. The guanine 1-imino Watson-Crick hydrogen-bonded protons, characteristic of the duplex state, are observed below 10°C, with solvent exchange occurring by transient opening of the tetranucleotide duplexes. The cytosine 4-amino Watson-Crick hydrogen-bonded protons resonate 1.5 ppm downfield from the exposed protons at the same position in the tetranucleotide duplexes, with slow exchange indicative of restricted rotation about the C-N bond below 15°C. The guanine 2-amino exchangeable protons in the tetranucleotide sequence exhibit very broad resonances at low temperatures and narrow average resonances above 20°C, corresponding to intermediate and fast rotation about the C-N bond, respectively. Solvent exchange is slower at the amino protons compared to the imino protons since the latter broaden out above 10°C. The well-resolved nonexchangeable base proton chemical shifts exhibit helix-coil transition midpoints between 37 and 42°C. The transition midpoints and the temperature dependence of the chemical shifts at low temperatures were utilized to differentiate between resonances located at the terminal and internal base pairs while the H-5 and H-6 doublets of individual cytosines were related by spin decoupling studies. For each tetranucleotide duplex, the cytosine H-5 resonances exhibit the largest chemical shift change associated with the helix-coil transition, a result predicted from calculations based on nearest-neighbor atomic diamagnetic anisotropy and ring current contributions for a B-DNA duplex. There is reasonable agreement between experimental and calculated chemical shift changes for the helix-coil transition at the internal base pairs but the experimental shifts exceed the calculated values at the terminal base pairs due to end-to-end aggregation at low temperatures. Since the guanine H-8 resonances of the CpCpGpG and d-CpCpGpG sequences exhibit upfield shifts of 0.6–0.8 and <0.1 ppm, respectively, on duplex formation, these RNA and DNA tetranucleotides with the same sequence must adopt different base-pair overlap geometries. The large chemical shift changes associated with duplex formation at the sugar H-1′ triplets are not detected at the other sugar protons and emphasize the contribution of the attached base at the 1′ position. The coupling sum between the H-1′ and the H-2′ and H-2″ protons equals 15–17 Hz at all four sugar rings for the d-CpCpGpG and d-GpGpCpC duplexes (25°C), consistent with a C-3′ exo sugar ring pucker for the deoxytetranucleotides in solution. The temperature dependent phosphate chemical shifts monitor changes in the ω,ω′ angles about the O-P backbone bonds, in contrast to the base-pair proton chemical shifts, which monitor stacking interactions.     

31P NMR Studies of the Binding of the Oligonucleotide (Ap)3A to an Oligodeoxythymidylate Covalently Linked to an Acridine Derivative

Abstract

³¹P NMR was used to study the specific interaction of an oligodeoxynucleotide containing four thymines and covalently attached to an acridine derivative through its 3-phosphate [(Tp)₄(CH₂)₅Acr] with a complementary oligoribonucleotide (Ap)₃A.³¹P-¹H and ¹H-¹H chemical shift correlation spectroscopies were jointly used to provide the assignment of the phosphorus resonances. A downfield shift of two phosphorus resonances of (Tp)₄(CH₂)₅Acr and of two phosphorus resonances of (Ap)₄A was observed upon complex formation. The assignment of the phosphorus resonances which are downfield shifted allowed us to propose a model involving an equilibrium between several 1:1 complexes where the acridine ring is intercalated between different A.T base pairs.     

NMR-based structure of anticancer drug mitoxantrone stacked with terminal base pair of DNA hexamer sequence d-(ATCGAT)2

Mitoxantrone is a promising antitumor drug having considerably reduced cardiotoxicity as compared to anthracyclines. Its binding to deoxyhexanucleotides sequence d-(ATCGAT)₂ has been studied by proton and phosphorous-31 nuclear magnetic resonance spectroscopy. The stoichiometry reveals that 1:1 and 2:1 mitoxantrone-d(ATCGAT)₂ complexes are formed in solution. Significant upfield shifts in 6H/7H, 2H/3H, 11NH, and 12NH protons (～.5?ppm) of mitoxantrone and T6NH imino protons (～.3?ppm) are observed. The phosphorous resonances do not shift significantly indicating that the base pairs do not open at any nucleotide step along the sequence of hexamer. Several inter-molecular Nuclear Overhauser Enhancement connectivities between mitoxantrone and hexanucleotide protons indicate that mitoxantrone chromophore stacks with terminal A1-T6 base pair and side chains involving 12CH₂, 12NH, and 14OH protons are in close proximity of A1, T2, A5, and T6 bases. Absorption and emission spectra show red shift in wavelength maxima, which is characteristic of stacking interaction. At higher mitoxantrone to nucleic acid ratios, electrostatic interactions are dominant. The 2:1 drug/DNA stoichiometric structure obtained by restrained Molecular Dynamics simulations shows considerable distortions in backbone torsional angles and helicoidal parameters although structural fluctuations in 25?ps analysis of trajectory are found to be negligible. Mitoxantrone binds as a monomer at either or both ends of hexamer externally with side chains interacting specifically with DNA. The findings are relevant to the understanding of pharmacological action of drug.     

Sequence Specific Molecular Recognition and Binding of a Monocationic Bis-Imidazole Lexitropsin to the Decadeoxyribonucleotide d-[(GATCCGTATG) (CATACGGATC)]: Structural and Dynamic Aspects of Intermolecular Exchange Studied by 1H-NMR

Abstract

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG) · (GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2′-endo-3′-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH₃(7). The base, anomeric H1′ and imino proton signals for the base sequence 5′-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5′-CCGT in the minor groove of the DNA NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the amino terminus oriented to C(4). The specific binding of 1 to the sequence 5′-CCGT-3′ deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci I fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA The [(T₇A₈T₉)· (A₁₂T₁₃A₁₄)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5′-CCGT sites, at a concentration of 12 mM, is estimated to be ～88s^?1 at 308°K with ΔG≠ of 63±5 K.J mol^?1.     

NMR Studies of a Lead Ribozyme and Its Non-Cleavable Analogue

Abstract

The structure of a lead ribozyme, which consists of two RNA strands, at neutral pH has been studied by NMR. Nearly all resonances of imino protons, base protons (H2, H5, H6 and H8) and sugar protons (H1′ and H2′) were assigned sequentially. Interesting structural features which deviate from the standard structure were found for the residues at an active site which consists of an internal loop. No indication of stable G:A base pairs was found in the loop. The effect of addition of Pb²⁺ was studied by the use of a non-cleavable analogue in which the cytidine at a cleavage site is replaced by 2′-O-methylcytidine. It was suggested that Pb²⁺ binds close to the cleavage site and that the structural change induced by Pb²⁺ is moderate and localized.

     

Multinuclear magnetic resonance studies of monomers and dimers containing 2'-fluoro-2'-deoxyadenosine

A series of 2′-fluorinated adenosine compounds, dAfl, dAflp, pdAfl, dAfl-A, A-dAfl, and dAfl-dAfl, have been investigated by nmr spectroscopies. The ¹H-, ¹⁹F-, and ³¹P-nmr data provide structural information from different parts of these moleucles. The pK_a of the phosphate group of these two 2′-fluoro-2′-deoxyadenosine monophosphates was found to be the same as that of hte parent adenosine monophosphate. As for the pentose conformation, the ³E population is greatly increased as a result of the fluorine substitution at the C_2′ position. However, the populations of conformers of gg (C_4′-C_5′) and g′g′ (C_5′-O_5′) and the average angle ?′(C_3′-O_3′) of the 2′-fluoro compounds remain unchanged as compared to the natural riboadenosine monomer and dimer (A-A). Thefefore, the backbone conformation of the 2′-fluoro-2′-deoxy-adenosine, its monophosphates and dimers, resembles that of RNA. The extent of base-base overlapping in these 2′-fluoro-2′-deoxy-adenosine-containing dimers is also found to be similar to or even greater than A-A. Thus, the conformations of these compounds can be considered as those in the RNA family. These fluorocompounds also serve as models for a careful study on the ¹⁹F-nmr in nucleic acid. The ¹⁹F chemical-shift values are sensitive to the environment of the fluorine atom such as ionic structure of the neighboring group(s) (phosphate of base), solvation, and ring-ruccent anisotropic effect from the base(s). Qualitatively, the change of the ¹⁹F chemical-shift values (up to 2 ppm) is much larger than that of ¹H-nmr (up to 0.5 ppm) in the dimers. Using dAfl·poly(U), poly(dAfl)·poly(dAfl), and poly(dAfl)·poly(U) helix–coil transition as model systems, the linewidth of ¹⁹F in dAfl- residues reflects effectively the mobility of the unit in the nucleic acid complex as calibrated by uv data and by ¹H-nmr. Therefore, application of ¹⁹F-nmr spectroscopy on fluorine-substituted nucleic acid can also be used to detect nucleic acid-nucleic acid interaction in complicated systems.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

A tetrameric allyl complex of sodium, and computational modeling of the Na-allyl chemical shift

Transmetallation of Li[A′] (A′ = [1,3-(SiMe₃)₂C₃H₃]⁻) with sodium tert-butoxide produces the corresponding sodium salt, which crystallizes from THF/toluene in the form of a cyclic tetramer, {Na[A′](thf)}₄. The Na atoms are in a square planar arrangement, bridged with π-bound allyl ligands; the Na-C distances range from 2.591(3)-2.896(3) Å, with an average of 2.70 Å. The geometries of several model organosodium complexes containing cyclopentadienyl and allyl ligands were optimized with density functional theory methods. The optimized structures were used with the gauge-including atomic orbital (GIAO) method to calculate their ²³Na NMR magnetic shielding values. Unlike the case with NaCp, the chemical shift of unsubstituted Na(C₃H₅) is very sensitive to the presence of coordinated THF (causing a 20 ppm upfield shift); silyl substitution has an even larger effect (30 ppm upfield shift). The observed ²³Na shift of δ −3.3 ppm for Na[A′] in THF-d₈, however, cannot be reliably distinguished from that calculated for the [Na(thf)₄]⁺ cation alone.     

2′,5′-Adenylate and Cordycepin Trimer Cores: Metabolic Stability and Evidence for Antimitogenesis without 5′-Rephosphorylation

Abstract

The effect of the 2′,5′-adenylate and cordycepin trimer cores on DNA and protein synthesis in human umbilical cord lymphocytes, lymphoblasts, peripheral blood lymphocytes and Epstein-Barr virus infected lymphocytes and their metabolism in tissue culture medium have been studied. [³²P]Adenylate and [³²P]- and [³H]cordycepin trimer cores were synthesized either enzymatically or chemically and added to cells in culture. The half-lives of the 2′,5′-A₃ core and 2′,5′-3′dA₃ core in tissue culture were 3 and 17 hr, respectively. Chromatographic analysis of the TCA-soluble extracts of the lymphocytes and lymphoblasts treated with 2′,5′-[³H]A₃ showed that 0.25% of the ³²P was identified as AMP, ADP, ATP and inorganic phosphate. By the more sensitive 2′,5′-p₃A₄[³²P]pCp radiobinding assay, 2′,5′-A₃ was detected in the TCA supernatants; however, there was no 5′-rephosphorylation. With the [³H]- and [³²P]cordycepin trimer core, 0.55% and 1.3% of the radioactivity was in the TCA soluble extracts. Although there was no 5′-rephosphorylation as determined by radiobinding assay, the intact cordycepin trimer core was detected by tlc, radiobinding assay, and HPLC.

Furthermore, in two experiments, the concentration of the cordycepin trimer core bound to or taken up by the lymphocytes was three-fold greater than the concentration in the medium. 2′,5′-A₃ and 2′,5′-3′dA₃ cores were both antimitogenic, but did not inhibit protein synthesis.