Periodate oxidation in chemistry of nucleic acid. Dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides

The employment of periodate oxidation in the chemistry of nucleic acids and their components is reviewed. The reaction mechanism, structural requirements to substrates, and synthesis of dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides are discussed in the first part. The second part involves chemical, physicochemical, and biological properties of the dialdehyde derivatives, as well as their use for the affinity modifications of proteins.     

Periodate oxidation in chemistry of nucleic acids: Dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides (Review)

The employment of periodate oxidation in the chemistry of nucleic acids and their components is reviewed. The reaction mechanism, structural requirements to substrates, and synthesis of dialdehyde derivatives of nucleosides, nucleotides, and oligonucleotides are discussed in the first part. The second part involves chemical, physico-chemical, and biological properties of the dialdehyde derivatives, as well as their use for the affinity modifications of proteins.     

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels-Alder Bioconjugation

Abstract

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

Novel method for the covalent immobilization of oligonucleotides via Diels-Alder bioconjugation

The synthesis of cyclohexadiene and maleimide derivatives and their use for the functionalization of oligonucleotides and the coating of glass surfaces is reported. A method for the covalent attachment of diene or maleimide modified oligonucleotides to the coated glass surfaces via aqueous Diels-Alder reactions is presented.     

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.     

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.     

SYNTHESIS OF OLIGONUCLEOTIDES BEARING AN ARYLAMINE MODIFICATION IN THE C8-POSITION OF 2′-DEOXYGUANOSINE

C8-Arylamine-dG adducts were converted into their corresponding 5′-O-DMTr-3′-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. The oligonucleotides were studied for their CD properties, T_m values, and their effects on primer extension assays using human DNA-polymerase β.     

Intraduplex Photo-cross-linking of p-Azidoaniline Residue and Amino Acid Side Chains Linked to the Complementary Oligonucleotides via a New Phosphorylating Intermediate Formed in the Mukaiyama System

Abstract

Oligonucleotide derivatives carrying a side chain of either lysine or histidine at the 3′-end and their complementary oligonucleotides having photoreactive groups a p-azidophenyl-NH(CH2)_nNH- (n = 4, 6) residue at the 5′-end were prepared by using new phosphorylating species formed by treatment of oligonucleotides with Ph3P and (PyS)₂ or (PyrS)₂. in DMF, DMSO or their mixture. Efficient cross-linking of duplexes occurred under UV-irradiation (λ > 300 nm).     

Synthesis of DNA-oligonucleotides damaged by arylamine-modified 2'-deoxyguanosine

C8-Arylamine-dG adducts bearing a labile N-formamidine group at the exocyclic amino function were converted into their corresponding 5'-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. These oligonucleotides were characterized by ESI-MS and enzymatic digestion and studied for their CD properties and Tm values.     

New light labile linker for solid phase synthesis of 2'-O-acetalester oligonucleotides and applications to siRNA prodrug development

We report on the synthesis and properties of oligonucleotides containing 2′-O-(levulinic acid) and 2′-O-(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we believe that these derivatives will have potential use as nucleic acid prodrugs. In addition, we report on the synthesis of a novel solid support with a photolabile linker that not only allows for the synthesis of oligonucleotides containing various 2′-O-acetalesters, but can be generally adopted to the synthesis of base-sensitive oligoribonucleotides. The release of oligonucleotides from this support is faster than with conventional linkers.     

Chemical Incorporation of 1-Methyladenosine,Minor tRNA Component,into Oligonucleotides

Abstract

The synthesis of suitably protected 1-methyladenosine derivatives has been developed and its successful chemical incorporation into oligonucleotides was achieved.     

Synthesis of steroid-containing oligonucleotides and their alkylating derivatives

New alkylating derivatives of oligonucleotides carrying a steroid (cholesterol, testosterone or ergosterol) residue have been synthesized, the residue being introduced via its hydroxyl group into the triester oligonucleotide block in the presence of triisopropylbenzenesulphonyl chloride and N-methylimidazole. Covalent attachment of steroids to oligonucleotides increases their hydrophobicity and does not influence the melting temperature of their complementary complexes. The data obtained showed that the oligonucleotide derivatives, bearing both an alkylating group of nitrogen mustard and a steroid residue, can be used as reagents for specific modification of nucleic acids.     

The effect of modifying terminal oligonucleotide linkages on their stability in cell cultures

The effect of modification of terminal groups of deoxyribooligonucleotides on their stability in cell culture and inside mammalian cells, namely Krebs 2, ascite carcinoma (KAC) and mouse fibroblasts L929, has been investigated. Oligonucleotides and their derivatives were found to be stable in culture medium without serum during 24 h. In the medium with KAC cells or ascitic fluid, orthophosphate was rapidly eliminated from the 5'-terminus of the oligonucleotides. In KAC cells, the scission of 5'-phosphomonoester bonds was accompanied by reutilization of the phosphate and by degradation of oligonucleotides to mononucleotides. In the medium with fibroblasts L929, the oligonucleotides were degraded from the 3'-end to tetranucleotides. Modification of oligonucleotides at the 5'-terminus by amidation made the 5'-phosphate groups resistant to KAC. Modification of the oligonucleotides by coupling of cholesterol or phenazinium to the 3'-terminus sufficiently increases their stability in the medium with fibroblasts L929, in that with Krebs 2 ascite carcinoma cells and inside the cells.     

Preparation of oligodeoxynucleotide 5'-triphosphates using solid support approach

We report a synthetic procedure for conversion of oligonucleotides to their 5'-triphosphate derivatives with moderate yield. The oligonucleotides were synthesized on solid support using standard phosphoramidite protocols. The DMT protection group was removed and the 5'-OH was phosphitylated using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one followed by reaction with tributyammonium pyrophosphate and iodine oxidation. After subsequent removal from support and complete deprotection, the products were isolated by anion-exchange HPLC chromatography. Structures of several 5'-triphosphate derivatives have been proven by phosphorus NMR, Mass-spectrometry and by HPLC comparison with authentic samples.     

Synthesis of oligonucleotides bearing an arylamine modification in the C8-position of 2'-deoxyguanosine

C8-Arylamine-dG adducts were converted into their corresponding 5-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. The oligonucleotides were studied for their CD properties, Tm values, and their effects on primer extension assays using human DNA-polymerase beta.     

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.     

Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.     

The synthesis of oligonucleotides containing an aliphatic amino group at the 5'' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.

A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated.     

Hydroxyl radical generation by oligonucleotide derivatives of anthracycline antibiotic and synthetic quinone.

For the first time the covalent binding of anticancer anthracycline drugs and their potential synthetic analogs to oligonucleotides of different sequences is proposed for obtaining site-specific DNA scission in systems in vitro and in vivo. New compounds such as daunomycin (Dm) and synthetic naphthoquinone (NQ), covalently bound to the heptadeoxynucleotide of pCCAAACA (Dm-pN7) and decadeoxythymidilate (pT10p-NQ), have been obtained. These oligonucleotide derivatives can form specific complexes with complementary oligonucleotide sequences; these compounds and their complementary complexes can be reduced by purified NADPH-cytochrome P-450 reductase. Using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), it has been shown that in aerobic conditions Dm-pN7 and pT10p-NQ are capable of generating OH radicals with and without complementary oligonucleotides. The chemical stability of the compounds in redox reactions has been studied. Oligonucleotide derivatives of natural and synthetic quinones capable of generating OH radicals seem to be a promising tool for site-specific scission of DNA in solution and in cells.