Fritz Eckstein

Nucleic acids and molecular biology

A wide range of topics are covered, including articles on nucleic acid structure, through their interactions with proteins to the control of gene expressions. A number of authors address the subject of RNA, including the difficult but important subject of its chemical synthesis, the complexities of its structures and the mechanisms of transcript splicing. The probing of DNA structure is reviewed in papers on the applications of dydroxyl radical and 1,10 phenanthroline copper cleavages. A number of important DNA-protein interactions are discussed, including DNA polymerase, the tryptophan and deoR repressors, and the resolvase enzymes which cleave Holliday junctions in recombination. Gene transcription is also covered, from the points of view of DNA methylation, mammalian ribosomal and avian lysozyme genes, and the control of transcription in the proto-oncogene c-fos. Finally, the plant kingdom has not been forgotten with articles on development and transposition in plants.

Phosphorothioates in molecular biology

The observation that phosphorothioate analogues of the nucleoside triphosphates are substrates for DNA- and RNA-polymerases has proven a boon for the molecular biologist. As these phosphorothioate-containing polymers are stable to degradation by nucleases and the sulfur atom confers many favourable chemical properties, several applications in molecular biology have been developed, including new methods for site-directed mutagenesis and DNA sequencing.

Chemically modified RNA: approaches and applications.

The modification of phosphate into phosphorothioate internucleotidic linkages in various RNAs and their usefulness in identifying phosphate positions essential for function are described. Several modifications of the 2′‐hydroxyl group of the ribose, particularly the replacement by fluorine atoms and amino groups, is discussed. These studies have been concentrated on hammerhead ribozymes in order to determine hydroxyl groups important for the catalytic activity. In addition these derivatives have been instrumental in rendering ribozymes more stable toward nucleases.—Heidenreich, O., Pieken, W., and Eckstein, F. Chemically modified RNA: approaches and applications. FASEB j 7: 90‐96; 1993.

Left-handed DNA: from synthetic polymers to chromosomes.

The interconversions between right-handed (R) and left-handed (L) helical conformations of DNA have been assessed by spectroscopic, electrophoretic, immunochemical, and enzymatic techniques. We have screened salt and solvent conditions which facilitate these transitions, as well as certain chemical modifications of the bases and backbone of defined synthetic polynucleotides. These include major and minor groove substituents as well as phosphorothioate analogues of selected phosphodiester bonds. We have established: R-L transitions in poly[d(G-C)] with iodo, bromo, methyl, and aza substitutions at the C5 position of cytosine, or phosphorothioate modification of the dGpC linkage. R-L transitions in the [d(A-C).d(G-T)]n sequence family using polymers modified as in the case of poly[d(G-C)]. The isomerizations are highly salt and temperature dependent. a possible L form of poly[d(A-T)] substituted with 2-amino adenine. the immunogenicities of constitutive and facultative Z-DNAs. the recognition specificities of different anti-Z-DNA IgGs for the spectrum of available polynucleotide probes. Some IgGs are sequence-specific. stabilization by IgG of otherwise transient left-handed conformations. anti-Z-DNA IgG binding to acid-fixed polytene chromosomes from the Diptera Drosophila, Chironomus, and Glyptotendipes. Laser scanning microscopy shows a maximal binding of 1 IgG per 3000-15,000 basepairs in acid fixed preparations. anti-Z-DNA IgG binding to negatively supercoiled plasmid, viral, phage, and recombinant closed circular DNAs. transcription from Z and Z* (associated) left-handed templates. From these and other results we propose that Z*-DNA may have important structural-functional roles in the cell.

Phosphorothioates, Essential Components of Therapeutic Oligonucleotides

Phosphorothioates have found their usefulness in the general area of oligonucleotide therapeutic applications. Initially this modification was introduced into the antisense methodology because of the nuclease resistance of the phosphorothioate linkage in comparison with that of the phosphate linkage. However, as experimental data accumulated, it was detected that this chemical modification also facilitates cellular uptake and bioavailibity in vivo. Thus, today the majority of therapeutic oligonucleotides contain this modification. This review will discuss the historical development of this modification and present some of its chemical properties where they differ from those of the phosphate group. The antisense application will be discussed in the original context with cleavage of the target mRNA, but other target RNAs such as microRNAs and long noncoding RNAs will also be covered. It continues with applications where the target RNA should not be cleaved. A brief presentation of decoy oligonucleotides will be included, as well as some miscellaneous applications. Cellular uptake is a crucial step for oligonucleotides to reach their target and will be briefly reviewed. Lastly, a most surprising recent observation is the presence of phosphorothioate groups in bacterial DNA where functions still remain to be fully determined.

The versatility of oligonucleotides as potential therapeutics

Oligonucleotides can in a variety of ways inhibit gene expression by interfering with translation. Oligonucleotides that are complementary to a target mRNA, antisense oligonucleotides, can prevent translation either by cleaving the target or by physically blocking the process. Additionally, oligonucleotides can correct the undesired splicing of pre-mRNA. RNA interference using double-stranded oligoribonucleotides also results in cleavage of the target mRNA. Catalytically competent ribozymes and DNAzymes can have the same effect. Even with no RNA as target, oligonucleotides can be selected as aptamers to bind to any protein to inhibit its activity. Moreover, oligonucleotides can act as decoys particularly for transcription factors to prevent binding to the promoter. A different mode of action is the activation of Toll-like receptors to induce an immune response. Several pathways for drug development are still in their infancy, for example microRNAs and antagomirs.

Human osteogenesis involves differentiation-dependent increases in the morphogenically active 3' alternative splicing variant of acetylcholinesterase.

ABSTRACT The extended human acetylcholinesterase (AChE) promoter contains many binding sites for osteogenic factors, including 1,25-(OH)2 vitamin D3 and 17β-estradiol. In differentiating osteosarcoma Saos-2 cells, both of these factors enhanced transcription of the AChE mRNA variant 3′ terminated with exon 6 (E6-AChE mRNA), which encodes the catalytically and morphogenically active E6-AChE isoform. In contrast, antisense oligodeoxynucleotide suppression of E6-AChE mRNA expression increased Saos-2 proliferation in a dose- and sequence-dependent manner. The antisense mechanism of action was most likely mediated by mRNA destruction or translational arrest, as cytochemical staining revealed reduction in AChE gene expression. In vivo, we found that E6-AChE mRNA levels rose following midgestation in normally differentiating, postproliferative fetal chondrocytes but not in the osteogenically impaired chondrocytes of dwarf fetuses with thanatophoric dysplasia. Taken together, these findings suggest morphogenic involvement of E6-AChE in the proliferation-differentiation balance characteristic of human osteogenesis.

INHIBITION OF GENE EXPRESSION WITH RIBOZYMES

Summary1. Ribozymes can be designed to cleavein trans, i.e. several substrate molecules can be turned over by one molecule of the catalytic RNA. Only small molecular weight ribozymes, or small ribozymes, are discussed in this review with particular emphasis on the hammerhead ribozyme as this has been most widely used for the inhibition of gene expression by cleavage of mRNAs.2. Cellular delivery of the ribozyme is of crucial importance for the success of inhibition of gene expression by this methodology. Two modes of delivery can be envisaged, endogenous and exogenous delivery. Of the former several variants exist, depending on the vector used. The latter is still in its infancy, even though chemical modification has rendered such ribozymes resistant against degradation by serum nucleases without impairment of catalytic efficiency.3. Various successful applications of ribozymes for the inhibition of gene expression are discussed, with particular emphasis on HIV1 and cancer targets. These examples demonstrate the promise of this methodology.

Designing ribozymes for the inhibition of gene expression

Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. However, particular attention must be paid to the following points: the identification of regions on the mRNA accessible to the ribozyme; the delivery of ribozymes to cells by either exogenous or endogenous delivery; colocalization of the ribozyme with the target RNA in the cell; and differentiation between closely related sequences. This field is advancing rapidly, and results obtained with transgenic animals demonstrate the power of this strategy for the inhibition of gene expression.

Isopolar vs Isosteric Phosphonate Analogues of Nucleotides

Abstract A range of β,γ-bridged phosphonate analogues of ATP and of β,β-bridged analogues of Ap4A has been synthesised. Some of their metal binding characteristics and inhibition of enzymatic phosphoryl transfer processes can be described in terms of the relative importance of steric and electronic features of the nucleotide analogues.