Interaction of cholesterol-conjugated alkylating oligonucleotide derivatives with cellular biopolymers.

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.     

Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment.     

Use of liposomes for incorporation of alkylating derivatives of mono- and oligonucleotides into mammalian cells]

It was shown that within the liposomes mono- and oligonucleotides and their alkylating derivatives penetrate the cells of Ehrlich ascite carcinoma and peritoneal exudate of the mice. Inside the cells the alkylating reagents are mainly utilized for modification of proteins (42--76%), RNA (5--16%) and DNA (3--9%). Presumably DNA modification is largely dependent on the penetration of the reagents into the nuclei. No significant differences in alkylation of the cell components by oligoadenylate derivatives, capable of complementary interactions with nucleic acids and mononucleotide derivatives, incapable of such interactions, were observed.     

Reactivity of oligonucleotide derivatives, containing a methylphosphate group. Affinity modification of target DNA by 4-N-(methyl-N-2-chlorethylamino)benzyl 3'- and 5'-phosphamide derivatives of octathymidylate containing methylphosphonate residues

Modification of 5'-32P-labelled octadecadeoxyribonucleotide d(pC5A8C5) (III) with octathymidylate methylphosphonate derivatives bearing both 3'- and 5'-terminal alkylating 4-(N-2-chloroethyl-N-methylamino)benzylphosphoamide residue has been investigated. Yield in the modification depends on configuration of methylphosphonate fragment, in case of Rp-isomer it may amount to 90%. Specificity of alkylation of nucleic acide target (III) by reagents based on the oligonucleotide methylphosphonates is almost the same as by reagents based on the oligonucleotides having phosphodiester internucleotide bonds.     

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.     

Inhibition of translation of immunoglobulin mRNA in vitro using an alkylating oligonucleotide derivative

Effect of complementary oligonucleotides and their reactive derivatives on translation of mouse immunoglobulin G kappa light chain was investigated. It was found that oligonucleotide pTGCTCTGGTTT and shorter oligonucleotides complementary to the coding sequence of the mRNA (nucleotides 205-215) do not arrest translation of the mRNA in the rabbit reticulocyte cell-free translation system. Preincubation of the mRNA with the alkylating 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide derivative of the oligonucleotide completely suppresses the synthesis of the protein thus demonstrating higher efficiency of the reactive oligonucleotide derivatives as inhibitors of the mRNA function.     

Special Properties of Modification of SsDNA Target by Alkylating Derivatives of Oligonucleotides in Tandem Complexes

Abstract

The influence of an effector (di-N-(2-hydroxyethyl)-phenazinium derivative of oligonucleotide) on modification of the DNA target by alkylating derivatives of oligonucleotides having various hybridization properties was studied. Being adjacent to the alkylating group of the reagent, the effector enhances the target modification if the oligonucleotide reagent has low hybridization properties and suppresses the modification if the reagent can form the stable complex with the DNA target at the used conditions.

     

Complementary addressed modification of single- and double-stranded DNA by alkylating derivatives of oligonucleotides isolated by partial DNA fragmentation

Reagents for complementary addressed modification of nucleic acids are proposed to be synthesized on the base of oligonucleotides obtained by partial chemical fragmentation of DNA. The alkylating 4-(N-2 chlorethyl-N-methylamino) benzyl-5'-phosphamide derivatives of 5'-[32P]-labelled oligonucleotides obtained from single and double-stranded DNA cloned in bacteriophage M13 mp9 have been synthesized. The alkylated derivatives of oligonucleotides selectively modify the complementary tracts of single-stranded DNA-target. They are also able to modify the complementary regions in double-stranded supercoiled plasmid DNA.     

A new strategy of discrimination of a point mutation by tandem of short oligonucleotides

A new strategy based on the use of cooperative tandems of short oligonucleotide derivatives (TSOD) has been proposed to discriminate a "right" DNA target from a target containing a single nucleotide discrepancy. Modification of a DNA target by oligodeoxyribonucleotide reagents was used to characterize their interaction in the perfect and mismatched complexes. It is possible to detect any nucleotide changes in the binding sites of the target with the short oligonucleotide reagent. In the presence of flanking di-3',5'-N-(2-hydroxyethyl)phenazinium derivatives of short oligonucleotides (effectors) the tetranucleotide alkylating reagent modifies DNA target efficiently and site-specifically only in the perfect complex and practically does not modify it in the mismatched complex. It has been shown that TSOD is much more sensitive tool for the detection of a point mutation in DNA as compared to a longer oligonucleotides.     

A procedure for selective DNA alkylation and detection by mass spectrometry.

A method which improves the detectability of DNA by mass spectrometry is presented. By quantitatively alkylating the backbone of phosphorothioate oligonucleotides the problems of gas phase ion generation by matrix assisted laser desorption ionization can be controlled. We have developed a selective alkylating protocol for phosphorothioate oligonucleotides which is a facile way of generating non-ionic nucleic acids. A variety of alkylating agents was studied and their kinetics were monitored in a gel electrophoretic assay and by mass spectrometry.     

Synthesis of oligonucleotide 2'-conjugates via amide bond formation in solution

An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide.     

Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.     

A New Strategy of Discrimination of a Point Mutation by Tandem of Short Oligonucleotides

Abstract

A new strategy based on the use of cooperative tandems of short oligonu-cleotide derivatives (TSOD) has been proposed to discriminate a “right” DNA target from a target containing a single nucleotide discrepancy. Modification of a DNA target by oligodeoxyribonucleotide reagents was used to characterize their interaction in the perfect and mismatched complexes. It is possible to detect any nucleotide changes in the binding sites of the target with the short oligonucleotide reagent. In the presence of flanking di-3′,5′-N-(2-hydroxyethyl)phenazinium derivatives of short oligonucleotides (effectors) the tetranucleotide alkylating reagent modifies DNA target efficiently and site-specifically only in the perfect complex and practically does not modify it in the mismatched complex. It has been shown that TSOD is much more sensitive tool for the detection of a point mutation in DNA as compared to a longer oligonucleotides.     

Suppression of Side Reactions During Final Deprotection Employing a Strong Acid in Boc Chemistry: Regeneration of Methionyl Residues from Their Sulfonium Salts

During global deprotection using a strong acid in Boc chemistry, the electrophilic alkylating species, i.e. carbocations, and formaldehyde generated from the side-chain protecting groups and the benzyloxymethyl group on the His residue, respectively, can cause alkylation of susceptible residues. To reduce these side reactions, a deprotection procedure using a strong acid such as HF or trifuloromethanesulfonic acid must always be carried out in the presence of scavengers. We found that addition of hydroxylamine derivatives could efficiently suppress the side reactions associated with formaldehyde and that addition of 2-mercaptopyridine could not only specifically circumvent alkylation of the Met residue but also convert its sulfonium salt to the Met residue regardless of the substituent species.     

Sequence-specific modification of nucleic acids by oligonucleotide derivative containing alkylating groups in the C-5-position of deoxyuridine]

A new type of alkylating derivatives of oligonucleotides with 4(N-methyl-N-2-chloroethylamino)benzyl (RCl) group at C-5 of deoxyuridine with a high extent of the target modification was prepared. The synthesized reagents d(ULNHRClCCACTT), where L = CH2 (Ia), CH2OCH2CH2 (Ib) and CH2NHCOCH2CH2 (Ic), proved to effectively (80-90%) modify the oligonucleotide d(TAAGTGGAGTTTGGC). The reagents (Ia) and (Ib) alkylate G6, G7 and G9 positions, while the reagent (Ic) modifies predominantly G9.     

Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells

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.     

Complementary addressed modification of plasmid DNA

The possibility to accomplish the sequence-specific chemical modification of superhelical DNA with reactive oligonucleotide derivatives was demonstrated. Plasmids containing fragments of the immunoglobulin gene were modified with alkylating derivatives of oligonucleotides complementary to a nucleotide sequence in the immunoglobulin gene. In contrast to the relaxed plasmid DNAs, superhelical DNAs (sigma = -0.1) were found to be attacked by the derivatives at the target nucleotide sequence. The efficiency of the reaction increases with the increase of the plasmids negative superhelicity. It was found also that the denatured derivatives. The sequence-specific modification of plasmid DNAs with the reactive oligonucleotide derivatives can be used for the site-directed mutagenesis and the investigation of the repair processes.     

Effect of the terminal phosphate derivatization of beta- and alpha-oligodeoxynucleotides on their antisense activity in protein biosynthesis, stability and uptake by eucaryotic cells.

Inhibition of polypeptide chain elongation with the mRNA-complementary (antisense) oligonucleotide has been realized through a RNase H independent mechanism. Nuclease resistant complementary non-natural alpha-17-mer oligonucleotide did not inhibit cell-free protein biosynthesis of beta-globin in the wheat germ system because it did not elicit RNase H activity. Linkage of alkylating group [4-(N-2-chloroethyl-N-methyl)-aminobenzyl]-methylamine to the 5'-terminus of the alpha-oligomer led to the formation of its covalent adduct with mRNA which could not be translated in vitro. Linkage of hydrophobic residues to the terminal phosphates of natural oligonucleotides increased their stability against nucleases and uptake by human cancer cells. A porphyrin, substituted in the meso-position by aromatic groups, gave a rise to an approximately six-fold increase of uptake and cholesterol a 30-100-fold increase. Eighty percent of bound derivatives were found in cytoplasmic cellular fractions.     

Synthesis, properties and interaction with eukaryotic cells of alkylating derivatives of oligodeoxyribonucleotides containing cholesterol or phenazinium residue covalently bound to the 3'-terminus

Radioactive alkylating 5'-[32P]-[4-(N-2-chlorethyl)N-methylaminobenzyl]-5'-phospham ide decadeoxyribothymidilate derivatives containing either free hydroxyl group (reagent I), hydrophobic cholesterol residue (reagent II) or polyaromatic phenazinium residue (reagent III) at 3'-termini were synthesized. The products were purified by HPLC and used for oligonucleotide-directed alkylating of DNA in isolated rat liver nuclei, Krebs-2 ascite carcinoma cells and L-929 murine fibroblasts. The uptake of reagent II by the cells was two orders of magnitude higher than that of reagent I and III. Intracellular alkylation of DNA by reagent II both in isolated nuclei and in living cells was about one order of magnitude higher than in the case of reagent I. The presence of phenazinium at 3'-termini of the reagent III leads to a sufficient increase of the alkylation extent compared to reagent I despite a quite low extent of its uptake by the cells.     

Interactions of derivatives of short oligonucleotides with nucleic acids. VII. Effect of conformation changes in the duplex structure on on the specificity and efficacy of modification of target DNA by alkylating oligonucleotide derivatives]

The modification of a target DNA by alkylating oligonucleotide derivatives possessing various capacities for complex formation was studied. The binding properties of oligonucleotides were changed either by increasing their length (tetra-, octa-, and dodecamers) or by introducing a point substitution and/or an N-(2-hydroxyethylphenazinium) residue. It was found that conformational changes occurring in the structure of the target.reagent complex upon elevating the reaction temperature affect the efficiency and site-specificity of the alkylation. In the case of complete saturation of the target with the reagent, an increase in the hybridization ability of the reagent reduced the efficiency of the target modification. It was found that the modification by the tetranucleotide reagent (in the presence of an effector adjacent to the 3' end) occurs exclusively at an intracomplex target base. In the case of the dodecamer, which forms a stable, highly cooperative complex with the target, several bases of the target undergo alkylation, and an increase in temperature changes the site-specificity of alkylation. In this process, the redistribution of the target modification sites toward stronger nucleophilic centers enhances alkylation at temperatures near the melting temperature of the target.dodecanucleotide complex despite a decrease in the extent of target association.