Subject(s)
Gene Expression Regulation/drug effects , Oligodeoxyribonucleotides, Antisense/chemical synthesis , Oligodeoxyribonucleotides, Antisense/pharmacology , Animals , Base Sequence , Biological Transport , Cell Line , Chromatography, High Pressure Liquid/methods , Drug Design , Genes, myc/drug effects , Oligodeoxyribonucleotides, Antisense/chemistry , Spectrometry, Mass, Secondary Ion/methods , Structure-Activity Relationship , ThionucleotidesABSTRACT
The use of chimeric oligonucleotides (ODN), in which certain phosphodiester internucleoside linkages are replaced by phosphorothioate (PS) linkages to provide protection against degradation by nucleases, is gaining increasing attention because of their significantly decreased propensity for nonantisense effects as compared with uniformly PS-modified ODN. We have recently reported that partially PS-modified ODN, in which end- capping is used to prevent hydrolysis by exonucleases in combination with PS protection at internal pyrimidine residues which are the major sites of endonuclease degradation, are surprisingly stable in serum. The present study investigates an additional role of the backbone modification in the stabilization of ODN against nucleolytic degradation. We show that the stability of an unmodified ODN in fetal bovine serum is significantly enhanced in the presence of PS-modified ODN. The magnitude of stabilization is strongly dependent on the type and degree of backbone modification. The observed effect is stronger for PS-modified ODN than for methylphosphonate (MP)-modified ODN and increases as the number of PS linkages in the ODN increases. Thus, nuclease stability of partially PS-modified ODN is not only caused by direct prevention of nuclease attack at the phosphate center but is additionally supported by interference of the nucleases with the PS groups of ODN, resulting in decreased degradation. As the degree of many nonantisense effects caused by ODN, such as protein interactions and B cell stimulation, is dependent on the backbone modification, our results may have implications for the use of non-ODN nuclease inhibitors to reduce undesirable side effects.
Subject(s)
Cell Nucleus/metabolism , Oligonucleotides, Antisense/pharmacokinetics , Animals , Base Sequence , Cattle , Chlorocebus aethiops , Drug Stability , Exonucleases/metabolism , Kinetics , Oligonucleotides, Antisense/blood , Pyrimidines , Thionucleotides , Vero CellsABSTRACT
The anti-herpes simplex virus type 1 (anti-HSV-1) efficacy of a series of oligonucleotides was determined as a function of their chemical structure. All oligonucleotides consisted of the same sequence directed against the translation initiation codon of the HSV-1 immediate early gene. The oligonucleotides were modified with phosphorothioate linkage patterns according to various protection strategies against nucleolytic degradation. We show that nuclease resistance is the dominant factor that determines the antiviral efficacy in this series. A minimal protection strategy, consisting of end-capping and pyrimidine protection, has proven to be particularly useful, because it not only yields nuclease-resistant oligonucleotides but also minimizes non-sequence-specific effects.
Subject(s)
Antiviral Agents/pharmacology , Deoxyribonucleases/metabolism , Herpesvirus 1, Human/drug effects , Oligodeoxyribonucleotides/pharmacology , Animals , Chlorocebus aethiops , Humans , Oligodeoxyribonucleotides/chemistry , Structure-Activity Relationship , Vero CellsABSTRACT
A subline of the human B cell lymphoma DHL-4, grown in the artificial serum-free medium HB101, displayed a resistant phenotype to the activity of an antisense oligodeoxynucleotide (aODN) effective on the parental DHL-4 line. It was found that the cellular uptake of the 18mer aODN in the two cell lines was almost the same. In contrast, the unresponsive subline DHL-4r degraded the aODN very efficiently, in contrast to the stability of aODN inside cells of the parental DHL-4 line. Activation of the degrading 'machinery' combined with selective properties of the artificial medium may be responsible for the loss of responsiveness to aODN.
Subject(s)
Antineoplastic Agents/pharmacology , Oligonucleotides, Antisense/pharmacology , Oligonucleotides/metabolism , Base Sequence , Drug Resistance , Humans , Lymphoma, B-Cell , Molecular Sequence Data , Oligonucleotides, Antisense/analysis , Phosphorus Radioisotopes , Tumor Cells, CulturedABSTRACT
Interactions of oligonucleotide derivatives with mammalian cells and cellular biopolymers have been investigated. The derivatives were oligonucleotides bearing an alkylating 2-chloroethylamino group at the 3'-end and a cholesterol residue at the 5'-terminal phosphate. These compounds are readily taken up by cells and react with cellular DNA, RNA and some proteins which may play a role in delivery of the compounds into cells.
Subject(s)
Biopolymers , Cholesterol/metabolism , Oligonucleotides/metabolism , Alkylation , Ascites/metabolism , Autoradiography , Cell Line , DNA/metabolism , Electrophoresis, Polyacrylamide Gel , Proteins/metabolism , RNA/metabolism , Tumor Cells, CulturedSubject(s)
Acholeplasma laidlawii/metabolism , Mycoplasma/metabolism , Oligodeoxyribonucleotides/metabolism , Oligonucleotides/metabolism , Animals , Biological Transport , Carcinoma, Krebs 2 , Culture Media , Drug Stability , Kinetics , L Cells , Mice , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
We have investigated the interaction of oligonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (less than 1 microM), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single- and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.
Subject(s)
Carcinoma, Krebs 2/metabolism , Oligodeoxyribonucleotides/metabolism , Receptors, Cell Surface/metabolism , Animals , Biological Transport , Carbon Radioisotopes , Electrophoresis, Polyacrylamide Gel , Kinetics , L Cells/metabolism , Mice , Molecular Weight , Receptors, Cell Surface/isolation & purification , Sucrose/metabolismABSTRACT
5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.
Subject(s)
Alkylating Agents/chemical synthesis , Cholesterol/pharmacology , DNA, Neoplasm/analysis , DNA-Binding Proteins/analysis , Oligonucleotides/chemical synthesis , Phenazines/pharmacology , Alkylating Agents/analysis , Alkylating Agents/pharmacology , Animals , Binding Sites , Carcinoma/analysis , Fibroblasts/analysis , Mice , Oligonucleotides/analysis , Oligonucleotides/pharmacologyABSTRACT
The alkylating derivatives of four individual diastereomers of the oligonucleotide [dTp(Et)]3dTpU and two individual diastereomers of oligonucleotide [dTp(Et)dTp]4 have been synthesized. The reagents with the phosphorus atoms in the enantiomeric p" configuration are shown to be more efficient in reacting with poly(dA) and with nucleic acids in Krebs-2 ascites carcinoma cells compared to those with the phosphorus atoms in the p' configuration.
Subject(s)
DNA/metabolism , Oligodeoxyribonucleotides/metabolism , Organophosphates/metabolism , Organophosphorus Compounds/metabolism , RNA/metabolism , Alkylation , Animals , Chemical Phenomena , Chemistry , Kinetics , Mice , Tumor Cells, CulturedABSTRACT
Sequence specific modification of nucleic acids with reactive oligonucleotide derivatives, complementary addressed modification, can provide an efficient approach for specific inactivation of certain cellular nucleic acids. In experiments with ascites tumor Krebs II cells and alkylating oligothymidylate derivatives it was found that alkylating oligonucleotide derivatives enter the living cell and modify complementary sequences in cellular nucleic acids with high efficiency. Complementary addressed modification of poly(A) sequences in cellular RNA with oligothymidylate derivatives was investigated in detail. The results of experiments on alkylation of cellular nucleic acids are consistent with complementary addressed modification of poly(A) sequences in cellular DNA. These results are supported by experiments on modification of chromatin DNA in which it was found that chromatin DNA interacts with oliogothymidylate derivatives more readily than the isolated double stranded DNA. It was found that alkylating oligonucleotide derivatives complementary to a sequence in immunoglobulin mRNA of MOPC 21 cells arrest the cellular immunoglobulin synthesis. Alkylating oligonucleotide derivatives complementary to RNAs of fowl plague virus inhibit virus multiplication in cell culture.