Biomedical subjects

C Altona

Publications and source records attributed to C Altona.

At least 55 records · Page 3Linked to original sources

PubMed1987

Conformational and model-building studies of the hairpin form of the mismatched DNA octamer d(m5C-G-m5C-G-T-G-m5C-G).

The hairpin form of the mismatched octamer d(m5C-G-m5C-G-T-G-m5C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3'-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5'-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5'-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual gamma' and beta+ torsion angles in residue dG(6). The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone. The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T-A-T4-T-A-G-G-A-T), in which the central -T4- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.

Base Composition↗

PubMed1986

The B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz two-dimensional NMR study.

The non-exchangeable proton resonances of the hexadeoxynucleoside pentakisphosphates d(m5C-G)3 and d(br5C-G)3 in the B form as well as in the Z form were assigned by means of two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy. The complete proton NMR spectrum of the B form of the methylated compound was assigned in a pure 2H2O solution as well as in a 2H2O/C2H3O2H mixed solvent, containing 5 mM MgCl2. In the latter solvent the B form occurs in slow equilibrium (on the NMR time scale) with the Z form, the resonances of which also were fully assigned. The proton resonances of the B and Z forms of the brominated fragment were assigned in a 2H2O/C2H3O2H solution containing 5 mM MgCl2. A new and general method is described for the sequential assignment of the non-exchangeable proton resonances of oligonucleotides in the Z form.

Bromine↗

PubMed1986

Conformational analysis of the B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz NMR study.

The B and the Z forms of the DNA hexamers d(m5C-G)3 and d(br5C-G)3 were investigated by means of NMR spectroscopy. It is demonstrated that the low-salt form of d(m5C-G)3 is a B DNA structure. The form, which becomes increasingly predominant when increasing amounts of MgCl2 and/or methanol are added to the solution, has Z DNA characteristics. It is shown that the major geometrical features of the Z form of d(m5C-G)3 in the crystal structure are maintained in solution, with the dC residues S sugar conformation, gamma + and the base in the anti orientation and the dG residues N (except the 3'-terminal residue), gamma t and syn. Neither the Z form of the methylated nor that of the brominated compound resembles the Z' form, in which the deoxy guanosine sugar rings adopt a C1'-exo conformation. Substitution of m5C by br5C causes no perceptible conformational changes in either the B or in the Z forms.

Bromine↗

PubMed1986

Conformational analysis of the octamer d(G-G-C-C-G-G-C-C) in aqueous solution. A one-dimensional and two-dimensional proton NMR study at 300 MHz and 500 MHz.

Proton NMR studies in H2O and 2H2O were carried out on the self-complementary DNA octamer d(G-G-C-C-G-G-C-C) and compared with similar studies on the dimethylated analogue d(G-G-m5C-m5C-G-G-C-C) [Sanderson, M. R., Mellema, J.-R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333-3346]. Base, H1', H2' and H2" resonances were assigned by means of two-dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature-dependent conformational behaviour and to extract thermodynamic parameters for the duplex-to-coil transition. Analysis of proton-proton couplings were used to discriminate between J1'2' and J1'2" and between the H2' and H2" resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B-DNA-type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration.

Carbohydrate Conformation↗

PubMed1986

Hairpin and duplex formation of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) in solution. An NMR study.

The partly self-complementary DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) was investigated by NMR spectroscopy in solution. It is demonstrated that this peculiar DNA fragment, under suitable conditions of concentration, salt and temperature, exclusively prefers to adopt a monomeric hairpin form with a stem of three Watson-Crick type base pairs and a loop of two residues. At high single strand concentration (8 mM DNA) and low temperature (i.e. below 295 K) the hairpin occurs in slow equilibrium with a B-dimer structure. At high ionic strength (greater than or equal to 100 mM Na+) and/or in the presence of methanol a third species appears, which is assigned to a Z-like dimer. In the B form, as well as in the Z dimer, the two central base pairs form G.T wobble pairs with the bases as major tautomers.

Base Sequence↗

PubMed1986

Influence of 2-aminoadenosine, A', on the conformational behaviour of d(T-A'-T-A'). A one-dimensional proton NMR study at 300 MHz and 500 MHz.

Proton NMR studies at 300 MHz and 500 MHz in aqueous solution were carried out on the modified self-complementary tetranucleoside trisphosphate d(T-A'-T-A'), in which d(A') represents 2-aminodeoxyadenosine. The NMR spectra were observed at two sample concentrations over the temperature range 2-70 degrees C. Assignments, based on homonuclear decoupling and nuclear Overhauser enhancement (NOE) experiments, are given. The concentration dependence of the chemical shift vs temperature profiles was used to extract information concerning duplex formation. The proton-proton and proton-phosphorus coupling constants were obtained at four temperatures and yielded accurate conformational data on the sugar ring and on the back-bone angles beta, gamma and delta. From the observed line broadening, shift profiles, NOEs, and from the observation of imino proton resonances, it is concluded that the compound exists as a miniduplex at low temperature. Comparison of these observations with similar observations on the parent compound d(T-A-T-A) indicate that substitution of dA by dA' increases the tendency towards duplex formation. At low temperature the compound adopts a stacked B-DNA type structure: destacking occurs on raising the temperature. The sequence-dependent sugar ring geometry [Mellema, J.-R., Pieters, J. M. L., van der Marel, G. A., van Boom, J. H., Haasnoot, C. A. G. & Altona, C. (1984) Eur. J. Biochem. 143, 285-301] is present in this molecule. The conformational parameters of d(T-A'-T-A') and d(T-A-T-A) are quite similar, thus substitution of dA by dA' has no measurable influence on the geometry of the sugar ring or on the backbone angles beta, gamma and delta.

Adenosine↗

PubMed1985

Conformational characteristics of the hexanucleoside pentaphosphate AUAUAU: a 2D NMR study at 500 MHz.

All 36 ribose proton resonances and most of the base proton resonances of the hexanucleoside pentaphosphate AUAUAU have been assigned unequivocally using 2D J-resolved spectroscopy, spin echo correlated spectroscopy (SECSY) and 2D NOE spectroscopy (NOESY). The NMR parameters of AUAUAU are compared with those of smaller fragments that contain methylated adenine bases: m62AU, m62AUm62A, m62AUm62AU and m62AUm62AUm62A. Previous studies on this series of compounds have shown that in all these cases purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as common feature that the interior pyrimidine residues are bulged out, whereas the purine residues stack upon each other. Chemical shift data, proton-proton coupling constants, as well as the observation of imino-proton resonances for AUAUAU show unambiguously that upon lowering the temperature the high-temperature "bulged out" situation reverts to a normal A-RNA-like double helix.

Computers↗

PubMed1985

A 1H and 31P NMR study of cis-Pt(NH3)2[d(CpGpG)-N7(2),N7(3)]. The influence of a 5'-terminal cytosine, on the structure of the cis-Pt(NH3)2[d(GpG)-N7,N7] intrastrand cross-link.

Proton NMR studies at 300 MHz and 500 MHz have been carried out on the trinucleoside bisphosphate d(CpGpG) and on cis-Pt(NH3)2[d(CpGpG)-N7(2),N7(3)] [abbreviated as d(CpGpGp) . cisPt]. For the Pt adduct, 13C and 31P NMR was also used for characterizing the oligonucleotide. d(CpGpG) appears to revert to a B-DNA-type single helix at lower temperatures. The relatively small concentration dependence of the proton chemical shifts, in comparison with shifts due to intramolecular stacking effects, indicates that the compound is essentially single-stranded. In d(CpGpGp) . cisPt, the first nucleoside, C(1), stacks well on top of the second, G(2), despite the N conformation of the G(2) sugar ring. The platinated GpG part in this trimer adopts largely the same structure as in cis-Pt(NH3)2[d(GpGpG)-N7(1),N7(2)] [den Hartog, J. H. J., et al. (1982) Nucleic Acids Res. 10, 4715-4730]. Main differences however, are changes in H8 chemical shifts and a 0.6-ppm downfield shift of the third nucleotide phosphorus, P(3), in d(CpGpGp) . cisPt with respect to P(2) in d(GpG) . cisPt. The latter shift change is likely to be induced by a structural alteration, caused by stacking of C(1) on top of G(2). Also, the large chemical shift differences between the two H8 protons in d(NpGpG) . cisPt fragments is discussed; the deviation from a mirror symmetry of the two guanine bases seems to be the main origin of this effect. The chemical shift changes, observed in the proton and phosphorus NMR chemical shift temperature and chemical shift pH profiles have been explained in terms of stack-destack equilibria changes.

Base Sequence↗

PubMed1985

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 4. The torsion angle distribution about the C3'-O3' bond in DNA constituents.

Carbon-13 and proton NMR spectra of a series of oligodeoxynucleotides (d(CT), d(CC), d(TA), d(AT), d(CG), d(GC), d(AG), d(AAA), d(TATA) and d(GGTAAT] were measured at various temperatures. The three coupling constants that are related to the magnitude of backbone angle epsilon (J(C4'-P), J(C2'-P) and J(H3'-P] are analyzed in terms of a three-state equilibrium about this bond. Two epsilon (trans) angles occur, which differ in magnitude depending on the conformation (N or S) of the adjoining deoxyribose ring. The S-type deoxyribose ring is associated with a smaller epsilon (trans) angle: epsilon (t,S) = 192 degrees. The N-type deoxyribose ring is associated with a larger epsilon (trans) angle epsilon (t,N) = 212 degrees. The third rotamer participating in the conformational equilibrium, is a gauche(-) (epsilon (-] conformer and occurs exclusively in combination with the S-type sugar ring (epsilon (-,S) = 266 degrees). Within the limits of experimental error, the magnitude of these three angles appears to be independent of the particular base sequence, except in the case of d(CG) where a slightly larger epsilon (t,S) angle (197 degrees) is indicated. A simple equation is proposed which may be used to calculate the population of epsilon (t,S) conformer in cases where only J(H3'-P) is known.

DNA↗

PubMed1985

cis-diamminedichloroplatinum(II) induced distortion of a single and double stranded deoxydecanucleosidenonaphosphate studied by nuclear magnetic resonance.

The structural distortion of a single- and a double-stranded decadeoxynucleotide upon binding of cis-PtCl2(NH3)2 was studied by 1H-NMR. After selective platination of d(T-C-T-C-G-G-T-C-T-C) (I) at the central d(-GpG-) site (resulting in I-Pt), several non-exchangeable base protons as well as H1', H2', H2" and H3' protons could be assigned by means of conventional NMR double-resonance techniques. Addition of the complementary decamer strand to I and I-Pt yielded the double-stranded III and III-Pt, respectively. All non-exchangeable base, H1', and most of the H2' and H2" protons in the two double stranded compounds could be assigned using 2D-chemical shift correlation (COSY) and nuclear Overhauser enhancement (NOESY) techniques. The double stranded compound III appears to adopt a B-DNA like structure. Comparison of NOEs and proton-proton coupling constants in the d(-GpG-).cisPt part in I-Pt and III-Pt reveals that their structure displays large similarity. Significant chemical shift changes (i.e. larger than 0.1 ppm) between III and III-Pt are restricted to the central four base pairs. It follows that the outer three base pairs, located on either side of the central four base pairs in III-Pt are likely to adopt a regular B-DNA type helix. The observed large upfield and downfield chemical shifts in the d(-CpGpG-) part of III with respect to III-Pt can be rationalized by describing the distortion of the double helix as a kink. A discussion of the observed physical effects upon platination of a double-stranded oligonucleotide is presented.

Base Sequence↗

PubMed1984

A phosphorus NMR study of the reaction products of cis-diamminedichloroplatinum (II) with a double-helical oligonucleotide and with DNA.

The structural distortion of oligonucleotides upon cis-PtCl2(NH3)2[d(T-C-T-C-G-G-T-C-T-C)-N7(5), N7(6)] reveals shifting of 4 phosphorus resonances due to platination. 3 Resonances could be assigned by selective 31P-irradiation, showing P(6) (P between the two Gs) to be shifted 1.5 ppm to low field. In the concomitant double strands P(6) is shifted 0.9 ppm to lower field. A similar peak has been observed in platinated salmon sperm DNA (37 degrees C), indicating that Pt-binding to GpG-fragments in DNA is similar to that found for the decanucleotide, so the distortion of DNA might be comparable.

Animals↗

PubMed1984

Sequence-dependent structural variation in single-helical DNA. Proton NMR studies of d(T-A-T-A) and d(A-T-A-T) in aqueous solution.

The two deoxyribotetranucleoside triphosphates d(T-A-T-A) and d(A-T-A-T) were investigated in aqueous solution by one- and two-dimensional proton NMR at 300 and 500 MHz. It is demonstrated that both compounds occur predominantly in the single-helical form. Accurate coupling constants are obtained by computer simulation of several 500-MHz spectra. The data are interpreted in terms of N and S pseudorotational ranges. The geometry of the major S-type conformers displays a clear sequence dependence, as expressed by variation of the endocyclic backbone angle delta (C5'-C4'-C3'-O3'). A simple sum rule is proposed to predict delta variation in single-helical DNA fragments. Comparisons are made with other sequence-dependent geometries as observed in a double-helical B-DNA fragment in the crystalline state. Furthermore, one- and two-dimensional nuclear Overhauser effect (NOE) spectroscopy was carried out on d(T-A-T-A). An inventory is made of the observed intra- and inter-residue NOEs. The NOE data confirm the presence of a highly stacked single-helical conformation of d(T-A-T-A) in solution. No indications are found for the formation of a bulge-out structure as observed for analogous alternating purine-pyrimidine oligoribonucleotides.

Base Sequence↗

PubMed1984

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 3. The magnitude of torsional angle epsilon in d(TpA) from CCOP and HCOP NMR coupling constants.

Carbon-13 NMR spectra of the deoxyribonucleotide d(TpA), 3',5'-cyclic AMP and 3',5'-cyclic dAMP were measured. It is shown that the different substitution of C2' in deoxyribonucleotides versus ribonucleotides does not affect the vicinal C2'-C3'-O3'-P coupling to a measurable extent. Therefore, the same set of Karplus parameters may be used for the C2'-C3-O3'-P couplings in ribonucleotides and in deoxyribonucleotides. Vicinal carbon-phosphorus and proton-phosphorus coupling constants are used to calculate the magnitude of the torsion angle epsilon (C4'-C3'-O3'-P), which amounts to 195(0) in the trans conformer and to 261(0) in the gauche(-) conformer.

Cyclic AMP↗

PubMed1984

Proton NMR study and conformational analysis of d(CGT), d(TCG) and d(CGTCG) in aqueous solution. The effect of a dangling thymidine and of a thymidine mismatch on DNA mini-duplexes.

Proton NMR studies of d(CGT), d(TCG) and d(CGTCG) were carried out at 300 and 500 MHz. The temperature and concentration dependence of the chemical shifts of various resonances indicates duplex formation only in the cases of d(TCG) and d(CGTCG). It is concluded that d(TCG) forms a mini-duplex stabilized by a 5'-dangling thymine base. Thermodynamic parameters of the duplex-to-coil equilibrium of the d(TCG) duplex are: delta H0 = -22.3 kcal/mol and delta S0 = -70 cal/mol. K, which correspond to approximately 40% duplex formation at 0 degrees C in a 2 mM nucleotide solution. Comparison of these data with thermodynamic parameters given earlier [Borer, P.N., Dengler, B., Tinoco, I. and Uhlenbeck, O.C. (1974) J. Mol. Biol. 86, 843-853] leads to the conclusion that the dangling base stabilization observed here is approximately equivalent to the stabilization caused by one or two additional A . T base pairs. The chemical shift behaviour of various resonances in d(CGTCG) indicates duplex formation without looping out of the thymine bases. The T X T mismatch does not seem to disturb the helical structure to a large extent. Analysis of the vicinal proton-proton coupling constants of the three compounds yielded geometrical data for the sugar rings. The data are interpreted in terms of N and S pseudorotational ranges. It is shown that a distinct conformation-transmission effect is exerted by the guanosine residues in a 5'----3' direction.

Magnetic Resonance Spectroscopy↗

PubMed1984

Conformational analysis of the single-helical DNA fragment d(T-A-A-T) in aqueous solution. The combined use of NMR proton chemical shifts and coupling constants obtained at 300 MHz and 500 MHz.

Proton NMR studies at 300 MHz and 500 MHz were carried out on the tetranucleoside trisphosphate d(T-A-A-T). The thermodynamics of the three stacking interactions, derived from chemical shift versus temperature profiles, were used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the fully stacked forms of the molecule. The data were interpreted in terms of N and S pseudorotational ranges [ Altona , C. and Sundaralingham , M. (1972) J. Am. Chem. Soc. 94, 8205-8212]. It is shown that the stacked state of the molecule cannot be described by one conformer, but consists of one major structure (60%) in which all sugar rings have S-type geometry and another structure (30%) in which residue dT(4) has an N-type sugar. The remainder of the stacked states consists of one or more conformers with two or three sugar residues in the N-type pseudorotational range. Detailed geometrical models are proposed for the major stacked conformers encountered in aqueous solution.

DNA, Single-Stranded↗

PubMed1984

Carbon-13 NMR in conformational analysis of nucleic acid fragments. 2. A reparametrization of the Karplus equation for vicinal NMR coupling constants in CCOP and HCOP fragments.

13C-31P coupling constants of 10 oligoribonucleoside phosphates, measured at a number of temperatures, are presented. The combination of these data with 1H-31P couplings of the same compounds leads to the derivation of two new and mutually consistent sets of Karplus parameters: J(CCOP) = 6.9cos2 phi--3.4cos phi + 0.7 J(HCOP) = 15.3cos2 phi--6.1cos phi + 1.6 At the same time new values for the base sequence dependent magnitude of the trans conformer of the backbone angle epsilon (C4'-C3'-O3'-P) are calculated. The present results show that the magnitude of epsilon(t) in right-handed ribo helices is confined to the range 214 degrees-226 degrees (average 219 degrees), which is in much better agreement with single crystal X-ray studies (average 218 degrees) than were previous deductions from NMR spectroscopic results (average 208 degrees).

Carbon Isotopes↗

PubMed1983

Conformational analysis of m4(2)C-m4(2)C-m6(2)A: a chemically modified 3'-acceptor end of tRNA, studied by NMR and CD methods.

A study on the conformation of the title compound, C-C-A, and on its constituent dinucleotides is presented. 1H-NMR spectra at 360 and 500 MHz were completely assigned by decoupling experiments. Computer simulation of the spectra yielded precise proton-proton and proton-phosphorus coupling constant values. The coupling constants are analyzed in terms of torsion angles and of N- and S-type sugar pucker. 31P-NMR spectra gave some information about P-O backbone torsion angles alpha and zeta. CD spectroscopy was used to obtain insight in the base-base interaction. The C(1) and C(2) unit in C-C-A show normal preference for N-type conformation of the sugar ring, whereas the A(3) residue appears rather biased towards the S-conformation. The zeta and alpha backbone torsion angles in the C-C phosphodiester linkage in C-C-A appear to assume normal g-, g- conformation, the zeta, alpha combination in the C-A linkage is proposed to have a g+, t conformation. In the C-C fragment in C-C-A a regular stack is indicated; it is suggested that the C-A part adopts an unusual antiparallel base stack.

Base Composition↗

PubMed1983

Carbon-13 NMR in conformational analysis of nucleic acid fragments. Heteronuclear chemical shift correlation spectroscopy of RNA constituents.

The assignment of the non-quaternary 13C resonances by means of two-dimensional heteronuclear chemical shift correlation spectroscopy is presented for several oligoribonucleotides: The dimers m6(2)AU, m6(2)Am6(2)A and mpUm6(2)A and the trimers m6(2)AUm6(2)A and m4(2)Cm4(2)Cm6(2)A. The temperature and concentration dependency of the 13C chemical shifts are studied with emphasis on the behaviour of the dimer m6(2)AU. The present study shows that in the 5-50 mM range the concentration-dependent chemical shift changes of the ribose carbons are negligible compared to chemical shift changes due to intramolecular events. All compounds studied show a surprising correlation between the chemical shifts of the carbon atoms of the ribose ring and the sugar conformational equilibrium as expressed by the percentage N or S conformer. Thus the chemical shift data can be used to obtain the thermodynamical parameters of the two-state N/S equilibrium. Parameters deduced for m6(2)AU are Tm = 306 K and delta S = -25 cal mol-1 K-1, which values are in satisfactory agreement with results obtained earlier from 1H NMR and from Circular Dichroism.

Dinucleoside Phosphates↗