Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy.

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments.

The effect of single base-pair mismatches on the duplex stability of d(T-A-T-T-A-A-T-A-T-C-A-A-G-T-T-G) . d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A).

The stabilitye and dynamics of the duplex d(T-A-T-T-A-A--T-A-T-C-A-A-G-T-T-G) . d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A) has been studied by means of ultraviolet-melting, temperature-jump relaxation kinetics, stopped-flow and NMR spectroscopy. In addition, the influence of the mismatches A .

Conformation of ribooligonucleotide duplexes containing an alternating C-G sequence which show an unusual circular dichroism spectrum.

The poly[r(G-C)] duplex shows an unusually large negative band in the long wavelength region of the CD spectrum. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized chemically and their properties were examined by UV and CD, and 1H and 31P NMR spectroscopy. These ribooligomers form a self-complementary duplex at low temperature, the CD spectrum of which shows a negative band at around 290 nm and a positive band at around 265 nm with almost equal magnitudes.

A one- and two-dimensional NMR study of the B to Z transition of (m5dC-dG)3 in methanolic solution.

The deoxyribose hexanucleoside pentaphosphate (m5dC-dG)3 has been studied by 500 MHz 1H NMR in D2O (0.1 M NaCl) and in D2O/deuterated methanol mixtures. Two conformations, in slow equilibrium on the NMR time scale, were detected in methanolic solution.

Gene A protein interacting with recombinant plasmid DNAs containing 25-30 b.p. of the phi X174 replication origin.

Synthetic oligodeoxyribonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the 30 b.

Oligonucleotide directed mutagenesis: selection of mutants by hemimethylation of GATC-sequences.

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically.

In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events.

Vasopressin and oxytocin mRNA regulation in the rat assessed by hybridization with synthetic oligonucleotides.

Vasopressin and oxytocin are nonapeptide hormones that regulate water metabolism and lactation, respectively. To study the regulation of the vasopressin and oxytocin genes at the mRNA level, we constructed a series of synthetic oligonucleotides, from 8 to 15 bases in length, for use in filter-blot hybridization assays (Northern blots) of hypothalamic mRNA levels and for primed synthesis of cDNAs from which we determined the nucleotide sequences of the 5' regions of the vasopressin and oxytocin mRNAs. A 20-fold increase occurred in the amounts of the two mRNAs present in the hypothalami of rats drinking 2% saline for three weeks.

Synthesis of short RNA fragments by the benzotriazolyl phosphotriester approach.

Analytical and experimental evidence will be presented to show that addition of a slight excess of 2-chlorophenyl-0,0-bis[1-benzotriazolyl]phosphate to a properly 2',5'-protected ribonucleoside gives an intermediate 3'-phosphotriester, which, after addition of a 2'-protected ribonucleoside, affords a dimer containing solely a 3'-5'-internucleotide linkage. The above phosphorylation procedure has been used in the synthesis of the pentamer UpApCpGpC.

Synthesis of ppTppp via phosphotriester intermediates.

The bifunctional and crystalline phosphorylating agent morpholino-0,0-bis[1-benzotriazolyl]phosphate has been used for the preparation of a 3',5'-bis-phosphotriester intermediate of thymidine. The latter has been converted into ppTppp by the following consecutive steps; removal of the benzotriazolyl group followed by the addition of phosphoric acid and removal of the 2-(4-nitrophenyl)-ethyl group followed by the addition of pyrophosphoric acid.

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.

Fourth rank immobile nucleic acid junctions.

An immobile nucleic acid junction composed of four dodecanucleotides has been designed according to principles of minimum symmetry aided by equilibrium calculations, and has been synthesized by automated phosphotriester techniques. We can demonstrate its tetrameric character and its 1:1:1:1 stoichiometry by gel electrophoresis. Thermal denaturation monitored by ultraviolet hyperchromism indicates that the complex is stable relative to its component arms.

Structure, kinetics and thermodynamics of DNA hairpin fragments in solution.

The hairpin-to-coil equilibrium of the hexadecadeoxynucleotide d(ATCCTATTTTTAGGAT) was extensively studied by means of NMR, T-jump and UV. The thermodynamic and kinetic parameters for this equilibrium were determined, yielding a consistent picture of the dynamical behavior of this hairpin structure, which is shown to be a clear example of a situation in which the linebroadening of the imino proton resonances is not determined by the lifetime of the double helix. A comparative study of the homologous hairpins in which the size of the loop was elongated from 4 to 7 thymidine residues shows a monotonous decrease in Tm for the hairpin-to-coil transitions.

Tandem repetition of the 5' mini-exon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription?

Activation of some variant surface glycoprotein (VSG) genes involves a duplicative transposition to an expression site, which completes the gene by addition of a mini-exon coding for the 5' 35 nucleotides of VSG mRNAs. Using a 22 nucleotide probe we have found some 200 copies of the mini-exon on a tandemly arranged 1.35 kb repetitive element.

Conformational analysis of a hybrid DNA-RNA double helical oligonucleotide in aqueous solution: d(CG)r(CG)d(CG) studied by 1D- and 2D-1H NMR spectroscopy.

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz1H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1', H2' and H2'' protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of 1H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Proton NMR studies on the covalently linked RNA-DNA hybrid r(GCG)d(TATACGC). Assignment of proton resonances by application of the nuclear Overhauser effect.

Proton NMR spectra of a covalently linked self-complementary RNA X DNA hybrid, r(GCG)-d(TATACGC), are recorded in H2O and D2O. Imino proton resonances as well as the non-exchangeable base and H-1' resonances are unambiguously assigned by means of nuclear. Overhauser effect measurements.

cis-Platinum induced distortions in DNA. Conformational analysis of d(GpCpG) and cis-pt(NH3)2[d(GpCpG)], studied by 500-MHz NMR.

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G-G chelated deoxytrinucleosidediphosphate platinum complex cis-Pt(NH3)2[d(GpCpG], on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis-Pt(NH3)2[d(Gp)]2, cis-Pt(NH3)2[d(pG)]2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis-Pt(NH3)2]2+. Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring.

3'-O-methylated analogs of 2-5A as inhibitors of virus replication.

We have explored analogs of 2'-5'-linked andeylic acid trimer (2-5A): 3'-O-methylated 2-5A, 2'-end modified adenylate trimer with deoxyadenosine or araadenine, methyl phosphonate and methyl phosphorotriester analogs as potential antiviral agents. For the treatment of virus infections, 2-5A and its analogs may serve in lieu of interferon, however, the use of 2-5A has two serious limitations: it is presumed to be impermeable to most cells, and moreover, cellular enzymes rapidly degrade it. Methylated analogs of 2-5A core strongly inhibited virus growth when added directly to cells in culture.

Conformational analysis of oligoarabinonucleotides. An NMR and CD study.

A 500 and 300 MHz proton NMR study of the series of oligoarabinonucleotides 5'aAMP, 3'aAMP, aA-aA, (aA-)2aA and (aA-)3aA is presented. In addition, circular dichroism is used to study the stacking behaviour of aA-aA. The complete 1H-NMR spectral assignment of the compounds (except the tetramer) is given.

Activation of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei.

We have studied the activation of genes for VSGs (variant surface glycoproteins) in Trypanosoma brucei (strain 427) in six independently isolated trypanosome clones; four expressing the gene for VSG 118 and two the gene for VSG 117. In all cases, gene activation is brought about by a duplicative transposition of the gene to an expression site located close to the end of a chromosome. The DNA segments flanking the expression-linked extra gene copy are nearly devoid of restriction enzyme recognition sites and their lengths vary by more than 10,000 base-pairs among different variants.

Assignment of non-exchangeable base proton and H1' resonances of a deoxyoctanucleoside heptaphosphate d(G-G-C*-C*-G-G-C-C) by using the nuclear Overhauser effect.

The resonances of the non-exchangeable base protons and 1' protons of the octamer d(G-G-C*-C*-G-G-C-C), C* = m5dC, have been assigned by means of NOE difference NMR spectroscopy at 500 MHz. From the measured J1'2' and J1'2" it follows that the octamer at low temperature prefers to adopt a B-DNA double-helical conformation in solution, however, some residual conformational freedom is detected at the 3' terminus. From the chemical shift versus temperature profiles it is concluded that no major conformational change occurs below 60-65 degrees C where the duplex formation for residues (2) to (6) is essentially completed under the conditions used.

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end.

Conformational analysis of a ribopentanucleoside tetraphosphate in aqueous solution. A two-dimensional NMR study at 500 MHz.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions.

A 500-MHz proton nuclear magnetic resonance study of the structure and structural alterations of gene-5 protein-oligo(deoxyadenylic acid) complexes.

The complex of the gene-5 protein of bacteriophage M13 with octadeoxyadenylic acid [d(A)8] has been shown earlier to differ in various respects from the complex with polynucleotides [Alma, N. C. M.

Conformational analysis of the single-stranded ribonucleic acid A-A-C-C. A one-dimensional and two-dimensional proton NMR study at 500 MHz.

Proton NMR studies at 300 MHz and 500 MHz are reported on the ribotetranucleotide A-A-C-C. The complete 1H-NMR spectral assignment at 20 degrees C is given. Two-dimensional NMR was used to elucidate spin multiplets in 'crowded' regions.