Biomedical subjects
Publications and source records attributed to R W Wagner.
Antisense oligodeoxynucleotides (ODNs) have great promise as agents for the specific manipulation of gene expression. Until recently, nonspecific effects of ODNs often confounded the interpretation of antisense studies. Improvements in ODN chemistry and cellular delivery techniques now allow for more potent and specific gene inhibition. This review critically evaluates recent progress in the development of antisense ODNs.
The binding of Rev to the Rev-response element (RRE) of the human immunodeficiency virus (HIV) is essential for RNA transport and expression of structural proteins such as gp160 encoded by env. To determine if env expression could be disrupted by complementary oligodeoxynucleotides (ODNs), band-shift studies were used to identify RRE sites that are essential for the formation of Rev-RRE complexes [Chin, D. J. (1992) J. Virol. 66, 600-607] or the stability of preformed complexes. In this report, we describe complete disruption of preformed Rev-RRE complexes by a subset of 15 ODNs complementary to stem-loop V. The most potent ODN complementary to bases CUGGGGCAUCAAGC disrupted 50% of preformed complexes at 1.2 microM, a 400-fold molar excess over the RNA. Expression of env in COS7 cells was blocked by nuclear microinjection of ODNs with C-5 propyne-modified pyrimidines and phosphorothioate linkages. Inhibition was highly dependent upon RNA target position, internucleotide chemistry, ODN sequence, and concentration. Unmodified phosphodiester or phosphorothioate ODNs were inactive. For the most potent ODN, 50% of the injected cells' env expression (I50) was blocked with 0.1 microM. A translational block is unlikely since these ODNs blocked expression of a luciferase vector in which the RRE was placed downstream of the termination codon. Consistent with their in vitro effects upon Rev-RRE complexes, stem-loop V ODNs were 9-fold more active than stem-loop II ODNs in blocking env expression while having a reduced (I50 = 0.27 microM) but equivalent potency against luciferase-RRE. These results suggest that disruption of Rev-RRE complexes may assist in blocking env expression.
A one flask synthesis of cis-substituted amphipathic porphyrins is reported. These porphyrins were used to study electrostatic effects on photoinduced electron transfer across the lipid bilayer-water interface. A neutral porphyrin undergoes only dynamic interfacial electron transfer reactions irrespective of charge of the acceptor, although ionic strength effects indicate a negative charge on the porphyrin donor species. A dianionic porphyrin forms an interfacial static complex with a dicationic electron acceptor, methyl viologen, at low ionic strength. The electron transfer rate within the complex is slow, 10(5) approximately 10(6) s-1, which is attributed to a near orthogonal orientation between the donor and the acceptor pi orbitals.
The intracellular distribution and metabolism of microinjected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, microinjected into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15-20 minutes. Microinjected fluorescent phosphorothioate ODNs remain in the nucleus for more than 24 hours. The persistence of fluorescence depends on the length of the ODN. Modification of the 3' end of phosphodiester ODNs does not significantly slow the rapid disappearance of fluorescence, although certain 3' modifications localize ODNs into the cytoplasm. Using specially designed ODNs, endonuclease activity is demonstrated to exist in the cytoplasm and nucleus. Modification of the 2' position of the ribose rings of a fluorescent phosphodiester oligodeoxynucleotide with O-methyl or O-allyl does not alter its intracellular distribution; however, the 2'-O-allyl modification stabilizes the persistence of fluorescence more than 60-fold compared to the 2'-deoxy control. Thus, the experiments indicate that somatic cells contain nucleolytic activities which degrade microinjected ODNs; however, chemical modification can dramatically circumvent this process.
Phosphorothioate oligodeoxynucleotides containing the C-5 propyne analogs of uridine and cytidine bind RNA with high affinity and are potent antisense inhibitors of gene expression. In a cellular assay, gene-specific antisense inhibition occurred at nanomolar concentrations of oligonucleotide, was dose-dependent and exquisitely sensitive to sequence mismatches, and was correlated with the melting temperature and length of oligonucleotide. Activity was independent of RNA target site and cell type but was detectable only when the oligonucleotides were microinjected or delivered with cell-permeabilizing agents. These oligonucleotides may have important applications in therapy and in studies of gene function.
We have used RNase H to study both the rates of oligonucleotide hybridization and dissociation at near-physiological conditions. We have studied the Effects of oligonucleotide length, mismatch, and chemical modifications on oligonucleotide association and dissociation with RNA. Dissociation results were compared with standard thermal melting curves to compare relative stabilities evaluated by the two techniques. Although generally the two techniques correlate for the compounds evaluated, we found several instances where the thermal melting curves failed to reflect the relative stability of different oligonucleotides at 37 degrees C using near-physiological conditions. This study suggests that direct measurement of hybridization and dissociation of an oligomer with RNA more accurately assesses the complicated kinetic scheme at 37 degrees C using near-physiological conditions than thermal melting curves would predict.
A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.
Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity that unwinds double-stranded RNA. We have now found that adenosine residues have been modified in the RNA products of this unwinding activity. Although the modified RNA remains double-stranded, the modification causes the RNA to be susceptible to single-strand-specific RNase and to migrate as a retarded smear on a native polyacrylamide electrophoresis gel. The modification is specific for double-stranded RNA. At least 40% of the adenosine residues can be modified in vitro in a given random sequence RNA molecule. By using standard two-dimensional TLC and HPLC analyses, the modified base has been identified as inosine. Mismatched base-pairing between inosine and uridine appears to be responsible for the observed characteristics of the unwound RNA. The biological significance of this modifying activity and also of the modified double-stranded RNA is discussed.
An RNA duplex unwindase activity has been found by using an in vitro assay with various types of mammalian, somatic cells, including HeLa, mouse plasmacytoma, and Burkitt lymphoma. The unwindase activity is very low in mouse fibroblast 3T3 cells arrested into quiescence, but increases when the cells are released into renewed growth by serum. In addition, a gel retardation assay proved to be specific and sensitive for detection of RNA duplex-unwindase complexes.
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The nuclease reactivity and specificity of a cloned tract of poly X (dA-dT) X poly(dA-dT) has been explored. Digestion with DNAse I, Mung Bean nuclease, S1 nuclease, DNAse II, and copper (1,10-phenanthroline)2 on a 256 base pair restriction fragment containing d(AT)14A revealed a dinucleotide repeat structure for the alternating sequence. Furthermore, conditions which wind or unwind the linear DNA had little effect on the reactivity of the AT insert. These preferred cleavages offer insights to structural alterations within the DNA helix which differ from A, B, or Z-DNA. Nucleation into flanking sequences by this structural alteration was not observed.
Platinum-pyrimidine complexes have been used as the sole electron-dense stains for electron microscopy on 18 types of mammalian cells. These recently discovered complexes react avidly with nucleic acids in vitro, are antitumor agents, and are highly soluble in water. In cells, they are selective for regions rich in nucleic acid and make visible the cellular chromatin, nucleolus, and ribosomes. In addition, cells that are tumorigenic exhibit electron-dense patches at the plasma membrane; normal cells do not. The results of treatment of ascites sarcoma-180 cells with concanavalin A, nucleases, glycosidases, or a protease suggest that the electron-dense patches are DNA, with neuroaminic acid involved in the surface binding.