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B Rayner

Publications and source records attributed to B Rayner.

67 records · Page 4Linked to original sources
PubMed1986

alpha-DNA. I. Synthesis, characterization by high field 1H-NMR, and base-pairing properties of the unnatural hexadeoxyribonucleotide alpha-[d(CpCpTpTpCpC)] with its complement beta-[d(GpGpApApGpG)].

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.

Chemical Phenomena↗
PubMed1986

Preparation of a short synthetic apurinic oligonucleotide.

The synthesis of the model apurinic oligonucleotide Tp(AP)pT is reported. Furthermore during the course of purification of this compound we have shown that the adduct formed upon reaction with methoxyamine has a Schiff base structure.

Apurinic Acid↗
PubMed1986

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.

Base Composition↗
PubMed1986

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.

Exons↗
PubMed1985

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.

Base Sequence↗
PubMed1984

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.

Base Sequence↗
PubMed1983

Use of inter-proton nuclear Overhauser effects to assign the nuclear magnetic resonance spectra of oligodeoxynucleotide and hybrid duplexes in aqueous solution.

Inter-proton nuclear Overhauser enhancements (NOEs) have been used to assign the aromatic, anomeric and 2' resonances in the 1H nuclear magnetic resonance spectrum of the duplex formed between d(T-C-A-C-A-T) and d(A-T-G-T-G-A). The same techniques have been applied to assignments in the hybrid duplex formed by d(T-C-A-C-A-T) with r(A-U-G-U-G-A). Comparison of intra-residue with inter-residue NOEs yields structural information which suggests that the conformations of both duplexes are similar. The NOEs are consistent with inter-proton distances measured from models of B-form DNA. Circular dichroic results confirm these deductions.

Chemical Phenomena↗
PubMedDate not supplied

Selective mRNA degradation by antisense oligonucleotide-2,5A chimeras: involvement of RNase H and RNase L.

Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives are currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. alpha-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of alpha-ON- and beta-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras.

Animals↗
PubMedDate not supplied

Esterase-triggered fluorescence of fluorogenic oligonucleotides.

In the prooligonucleotide approach, a step of activation by cellular esterases is necessary for the removal of internucleoside phosphate masking groups and subsequent intracellular delivery of active antisense oligonucleotides. The efficacy of this approach implies that prooligonucleotides, once they are taken up by cells, are demasked by esterases during their course to their nucleic acid targets. In this regard, a method for labeling oligomers with esterase-activable fluorogenic tag was designed. The two phenolic functions of carboxyfluorescein were protected by pivaloyl groups, yielding a nonfluorescent lactone which was further activated as a N-hydroxysuccinimide ester. Two nuclease-resistant phosphorothioate 18-mer and methylphosphonate 19-mer oligodeoxynucleosides were attached to this biprotected fluorescein derivative via an amino linker at the 5'-end of the oligomers. The two conjugates were assayed for their carboxyesterase substrate ability in different biological media. In the presence of purified esterases or when incubated in serum or cell extracts, both oligonucleotide conjugates became fluorescent. In addition, the phosphorothioate oligoconjugate was microinjected into the cytoplasm of human fibroblasts, and a fast cytoplasmic release of fluorescence was observed with a rapid translocation of the fluorescent oligomer into the nucleus.

Ammonia↗
PubMedDate not supplied

Prooligonucleotides exhibit less serum-protein binding than phosphodiester and phosphorothioate oligonucleotides.

The protein-binding properties of dodecathymidine derivatives (prooligos) bearing either methyl- or tert-butyl-S-acylthioethyl (Me- or tBuSATE) protecting groups were evaluated. The dissociation constants (Kd) were estimated for complexes of prooligos with serum blood proteins and lactoferrin using prooligos to compete the binding of the radiolabeled, alkylating probe oligonucleotide CIRp(T)12 with the proteins. tBuSATE and MeSATE prooligos have decreased affinity of binding with serum proteins and lactoferrin compared with their parent oligos. These data suggest that prooligos should cause less side effects which combined with their stability to nucleases and enhanced permeability into cells make them potentially useful for design of therapeutics.

Animals↗