Oligonucleotide derivatives bearing hemin and deuterohemin groups were synthesized. The derivatives efficiently react with the complementary nucleotide sequence in ssDNA forming covalent adducts and piperidine-labile sites. In the case of the deuterohemin derivative, some direct cleavage of the target DNA occurs.

Base Sequence↗

PubMed1990

Sequence-specific chemical modification of chromatin DNA with reactive derivatives of oligonucleotides.

Chemical modification of the chromatin DNA with alkylating derivatives of oligothymidylate (pT)16 and oligoadenylate (pA)16 bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group at the 5'-phosphate has been investigated. It was found that the derivatives do react with DNA in chromatin. The reactions occur presumably at the complementary sequences of the DNA since the reaction of the oligothymidylate derivative is inhibited by oligonucleotide (pT)16 taken in excess and is not influenced by hexadecanucleotide of a random structure. Isolated DNA does not react with the oligothymidylate derivative. It is concluded that in chromatin, DNA is partially unwound or possesses some sites which can be opened easily in the presence of complementary oligonucleotides.

Alkylating Agents↗

PubMed1990

Antisense oligonucleotide derivatives as gene-targeted drugs.

The strategies and problems involved in designing oligonucleotide derivatives as gene-targeted drugs are discussed. Experiments with isolated and cellular nucleic acids, studies with infected cell cultures, and preliminary animal tests all demonstrate that various derivatives of complementary oligonucleotides (antisense oligonucleotide derivatives) can act as extremely specific and potent inhibitors of gene expression. The design and synthesis of more stable oligonucleotide analogues that can enter mammalian cells and efficiently affect preselected nucleic acids will result in the development of a new generation of drugs, including those with antiviral and anticancer properties.

Animals↗

PubMed1989

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.

Alkylating Agents↗

PubMed1989

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

Affinity Labels↗

PubMed1989

Mechanism of oligonucleotide uptake by cells: involvement of specific receptors?

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.

Animals↗

PubMed1988

Sequence-specific chemical modification of double-stranded DNA with alkylating oligodeoxyribonucleotide derivatives.

Chemical modification of double-stranded (ds) DNA with alkylating oligodeoxynucleotide (oligo) derivatives, 5'-p(N-2-chloroethyl-N-methylamino) benzylamides of oligos, has been investigated. In contrast to relaxed plasmid DNAs, the superhelical molecules interact with the oligo derivatives and specific alkylation of the DNAs occurs at the regions complementary to the oligo reagents. Alkylating derivatives of oligocytidylates and pT(pCpT)6 react with corresponding homopyrimidine-homopurine tracts within ds DNA fragments due to triple helix formation.

Alkylation↗

PubMed1988

Site-specific cleavage of single-stranded DNAs at unique sites by a copper-dependent redox reaction.

Metal ions play a crucial role not only in the formation and maintenance of nucleic acid structure, but also in important biochemical conversions of polynucleotides. Some aqueous metal ions, acting as general acid/base (or electrophilic/nucleophilic) catalysts, can induce site-specific cleavage of RNA. DNA is not cleaved efficiently by the non-redox metal-induced mechanism. However, DNA degradation by radicals formed in the metal-catalysed auto-oxidation of ascorbate (or other reducing agents) is well known. In the past, the observed cleavage reactions have not been very specific. Here, we report a non-enzymatic cleavage of single-stranded DNA occurring at unique sites due to redox reactions involving copper. This could be considered a 'self-cleavage' reaction, by analogy with the lead-induced non-redox RNA cleavage reaction. This site-specific cleavage of DNA, stimulated by ascorbate and hydrogen peroxide, is most efficient under physiological conditions, so this phenomenon may have biological significance.

Ascorbic Acid↗

PubMed1988

Complementary addressed modification of nucleic acids with the alkylating derivatives of oligothymidylate ethyl phosphotriesters. Effect of the phosphotriester fragments' configuration.

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.

Alkylation↗

PubMed1988

Sequence-specific chemical modification of a hybrid bacteriophage M13 single-stranded DNA by alkylating oligonucleotide derivatives.

Alkylating oligonucleotide derivatives react with the complementary sequences in hybrid M13mp7 bacteriophage single-stranded DNA and destroy the infecting ability of the DNA. The reagents do not damage M13mp9 single-stranded DNA lacking the target nucleotide sequence.

Alkylation↗

PubMed1986

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.

Alkylation↗

PubMed1985

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea.

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.

Alkylation↗

PubMed1985

Sequence-specific chemical modification of a 365-nucleotide-long DNA fragment with an alkylating oligonucleotide derivative.

An aromatic 2-chloroethylamino group was attached to the 5'-terminal phosphate of the oligodeoxyribonucleotide pCCCTCTTTCTT. The oligonucleotide derivative prepared was used for modification of the 365-nucleotide-long DNA fragment. It was found that modification of the fragment proceeds in a sequence-specific way at 3 guanosine residues within the sequence complementary to the oligonucleotide reagent.

Alkylation↗