Crystal structure of a Z-DNA fragment containing thymine/2-aminoadenine base pairs.

The Z-DNA structure has been shown to form in two crystals made from self-complementary DNA hexamers d(CGTDCG) and d(CDCGTG) which contain thymine/2-aminoadenine (TD) base pairs. The latter structure has been solved and refined to 1.3 A resolution and it shows only small conformational changes due to the introduction of the TD base pairs in comparison with the structure of d(CG)3.

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.

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.

Use of a 1-hydroxybenzotriazole activated phosphorylating reagent towards the synthesis of short RNA fragments in solution.

Evidence will be presented to show that the reagents 3a-c (X = O) obtained by the reaction of 2-chlorophenylphosphorodichloridate with 1-hydroxybenzotriazole or 1-hydroxy-6-trifluoromethyl (6-nitro) benzotriazoles, respectively, do not give, under normal coupling conditions, phosphorylation of the lactam functions in uracil or guanine. Reagent 3b (X = O) proved to be very convenient for the in situ formation of 3'-5'-phosphotriester linkage. The latter will be illustrated by the synthesis of several RNA fragments.

Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution.

UV and CD data of the partially self-complementary heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG), obtained as a function of temperature, salt and strand concentration, show that: at low NaCl and strand concentration the oligomer exhibits, on increasing the temperature, a biphasic thermal profile which is indicative of two structural transitions, from dimeric duplex to hairpin and from hairpin to coil; the loop stabilizes enthalpically both B and Z hairpin structures with respect to the corresponding unconstrained hexamer d(CGCGCG) by a few Kcal/mol; the oligomer undergoes a B-Z transition which appears to be complete, at 0 degree C, when induced by NaClO4; by contrast the B-Z transition induced by NaCl does not reach completeness even at salt saturation. The independence of the denaturation temperature, at high salt conditions, on the oligomer concentration indicates that the Z structure is present also in the hairpin.

Non-Watson-Crick G.C and A.T base pairs in a DNA-antibiotic complex.

The structure of a DNA octamer d(GCGTACGC) cocrystallized with the bisintercalator antibiotic triostin A has been solved. The DNA forms an unwound right-handed double helix. Four base pairs are of the Watson-Crick type while four are Hoogsteen base pairs, including two A.

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.

On loop folding in nucleic acid hairpin-type structures.

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations.

Synthesis of nucleic acid methylphosphonates via the 1-hydroxybenzotriazole phosphotriester approach.

Dinucleoside methylphosphonates can easily be prepared starting from properly protected d-nucleosides and the bifunctional phosphorylating reagent methyl-O,O-bis(1-benzotriazolyl)phosphate. Separation of the diastereoisomers of 5'-DMTR-d-Ap(Me)T-3'-lev affords optically pure dinucleoside methylphosphonates which, after removal of the 3'-levulinoyl group, have been used for the synthesis of the two optically pure diastereoisomers of the hexamer d-CpGpAp(Me)TpCpG. Further, a one-pot procedure for the preparation of uridine-3',5'-cyclic methylphosphonate will be described.

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.

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC).

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2.

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs.

Z-DNA forms without an alternating purine-pyrimidine sequence in solution.

Nuclear magnetic resonance spectra (proton and phosphorus-31) and ultraviolet absorption spectra of the DNA decamer d(br5CGbr5CGATbr5CGbr5CG), in which the central two adenine-thymine base pairs are out of order with the rest of the purine-pyrimidine alternation sequence, indicate that under appropriate solvent conditions (high salt and methanol) the molecule undergoes a structural transition from a right-handed B-DNA conformation to a left-handed Z-DNA conformation. Measurements of the two-dimensional nuclear Overhauser effect on the decamer indicate that all of the guanines as well as the two equivalent thymines adopt the syn conformation.

Investigation and treatment of renal calculi.

We have investigated and treated 176 patients who were suffering from renal calculi. The stones contained calcium in 87% of patients, predominantly urate in 11%, and rarely contained magnesium ammonium phosphate or cystine. Of the patients with calcium stones, hypercalciuria was present in 75% and was identified in 57% by the measurement of the 24-hour urinary calcium excretion, and in a further 18% by a standardization calcium "fast-and-load" test.

Hairpin formation in synthetic oligonucleotides.

The structure and dynamics of the homologous series of the (partly) self-complementary DNA fragments, d(ATCCTATnTAGGAT) n = 0-7, were investigated in a combined NMR, T-jump, and optical melting study. It is shown that all compounds in the series may adopt hairpin like conformations, even for n less than 3, although for these smaller n values this only occurs in significant amounts at relatively low concentrations (approximately 10 microM). The enthalpy change accompanying the hairpin-coil melting transition turns out to depend on the number of intervening thymidines, n.

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.

Crystal structure of Z-DNA without an alternating purine-pyrimidine sequence.

In left-handed Z-DNA, consecutive nucleotides along the chain alternate in the syn and anti conformations. Purine residues form the syn conformation readily and up to now all Z-DNA crystal structures have sequences of alternating purines and pyrimidines. However, we find that d(C-G-A-T-C-G) with the cytosines brominated or methylated on C-5 crystallizes as Z-DNA.

Synthesis of riboguanosine pentaphosphate ppprGpp (Magic Spot II) via a phosphotriester approach.

The bifunctional phosphorylating reagent O,O-bis[1-benzotriazolyl]phosphoromorpholidate was used to introduce a 5'-O-triphosphate and a 3'-O-diphosphate function in a partially protected riboguanosine. Pilot studies indicated that protection of the 2'-OH of ribonucleosides with the acid-labile tetrahydropyranyl group, instead of the more labile 4-methoxytetrahydropyran-4-yl group, was most satisfactory for the preparation of the Magic Spot II.

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.

A comparison of the structure of echinomycin and triostin A complexed to a DNA fragment.

Two members of the quinoxaline antibiotic family, echinomycin and triostin A, form crystals complexed to a DNA fragment with the sequence d(CpGpTpApCpG). The crystal structure of both complexes was solved by X-ray diffraction to near-atomic resolution. The two structures are similar to each other with differences in some details due to the shorter cross bridge of echinomycin.

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.

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.

Structural basis for stabilization of Z-DNA by cobalt hexaammine and magnesium cations.

In the equilibrium between B-DNA and Z-DNA in poly(dC-dG), the [Co(NH3)6]3+ ion stabilizes the Z form 4 orders of magnitude more effectively than the Mg2+ ion. The structural basis of this difference is revealed in Z-DNA crystal structures of d(CpGpCpGpCpG) stabilized by either Na+/Mg2+ or Na+/Mg2+ plus [Co(NH3)6]3+. The crystals diffract X-rays to high resolution, and the structures were refined at 1.

Synthesis of phosphorothioate-containing DNA fragments by a modified hydroxybenzotriazole phosphotriester approach.

The phosphorothioylating agent which was obtained by treating 2,5-dichlorophenyl phosphorodichloridothioate with 1-hydroxy-6-nitrobenzotriazole proved to be very effective for the synthesis in solution and on a solid support of phosphorothioate-containing DNA fragments.