Biomedical subjects

Dmitry A Stetsenko

Publications and source records attributed to Dmitry A Stetsenko.

14 recordsLinked to original sources

PubMed2005

Oligonucleotides containing 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine as suitable precursors of 2'-aldehyde oligonucleotides for chemoselective ligation.

2'-O-[2-(2,3-Diacetoxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite was prepared and used in solid-phase synthesis to obtain oligonucleotides containing a 1,2-diol group, which may then be converted into a 2'-aldehyde group. The oligonucleotides were conjugated efficiently to various molecules by chemoselective ligation that involves an addition-elimination reaction between the 2'-aldehyde group and a suitable nucleophile, such as a hydrazine, a O-alkylhydroxylamine or an 1,2-aminothiol. The method was applied successfully to the conjugation of peptides to oligonucleotides at the 2'-position.

Aldehydes↗

PubMed2005

Antiviral activity of steric-block oligonucleotides targeting the HIV-1 trans-activation response and packaging signal stem-loop RNAs.

Mixmer oligonucleotides consisting of residues of both 2'-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5'-UTR of HIV-1 RNA, the transactivation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR beta-Gal cells and subsequently infected with HIV-1.

5' Untranslated Regions↗

PubMed2005

2'-Hydrazine oligonucleotides: synthesis and efficient conjugation with aldehydes.

Oligodeoxyribonucleotides that contain a novel nucleoside, 2'-O-(2-hydrazinoethyl)uridine, were synthesized by sodium cyanoborohydride reduction of hydrazones formed from 2'-aldehyde oligonucleotides and FmocNHNH2 followed by ammonia deprotection. 2'-Hydrazine oligonucleotides were used successfully to obtain a series of conjugates with various aldehydes by hydrazone formation. The method was shown to be suitable for the preparation of oligonucleotide-peptide conjugates.

Aldehydes↗

PubMed2005

Incorporation of side-chain groups of basic amino acids into oligonucleotides via the 2'-position of uridine.

Certain amino acids and short peptides are known to act as enhancers of ribozyme-mediated RNA cleavage at low concentration of magnesium ion. Thus, covalent conjugates of oligonucleotides and some amino acids may have a potential for development as sequence-specific artificial ribonucleases. Here we would like to report an incorporation of basic amino acids, lysine and histidine, into oligonucleotides at the 2'-position of a uridine residue. The approach involves the 2'-O-alkylation and attachment of the corresponding amino acid by the alpha-amino group through a urethane-type linker.

Amino Acids, Basic↗

PubMed2005

Chemical methods for peptide-oligonucleotide conjugate synthesis.

Methods of peptide-oligonucleotide conjugate synthesis are presented that may be useful in the study of cell targeting and delivery of oligonucleotides and their analogs. The first method involves total stepwise solid-phase synthesis on a single support. The second involves preparation of oligonucleotides containing 2'-aldehydes and subsequent chemoselective ligation with functionalized peptides to form hydrazine, oxime, or thiazolidine linkages.

Oligonucleotides↗

PubMed2004

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.

Amides↗

PubMed2003

Pyrenemethyl ara-uridine-2'-carbamate: a strong interstrand excimer in the major groove of a DNA duplex.

The synthesis of new nucleoside derivatives, ara-uridine-2'-carbamates, and their incorporation into synthetic DNA oligomers is described. The modification directs ligands into the major groove of duplex DNA and somewhat destabilizes the duplexes of modified oligonucleotides with complementary DNA or RNA. In the case of pyrenemethyl carbamate modification in DNA-DNA duplexes, the destabilization is considerably reduced. The pyrenemethyl derivative also shows remarkable spectral properties: a "reversed" absorbance change for pyrene at 350 nm in the course of denaturation of the DNA duplex, as compared to the change seen in the nucleotide absorbance at 260 nm. This derivatization also causes pronounced sequence-dependent excimer formation in the major groove.

DNA↗

PubMed2003

A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2'-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2'-thio-LNA residues.

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

Calcitriol↗

PubMed2002

(R)-2,4-Dihydroxybutyramide seco-pseudonucleosides: new versatile homochiral synthons for synthesis of modified oligonucleotides.

[reaction: see text] Two series of seco-pseudonucleoside synthons were synthesized from (R)-(+)-alpha-hydroxy-gamma-butyrolactone and (R)-(-)-pantolactone by aminolysis, side-chain protection, dimethoxytritylation, and phosphitylation or solid-phase attachment. The phosphoramidites and solid supports were used in automated DNA synthesis to prepare oligonucleotides modified with one or more 2,4-dihydroxybutyramide units bearing side-chain reporter groups. These new oligonucleotide modification reagents allow the introduction of a label into any desired position within an oligonucleotide chain during solid-phase assembly.

Amides↗

PubMed2002

Total stepwise solid-phase synthesis of oligonucleotide-(3'-->N)-peptide conjugates.

[structure: see text] An efficient total stepwise solid-phase synthesis of oligonucleotide-(3'-->N)-peptide conjugates is described that makes use of either a controlled pore glass support or macroporous polystyrene beads. Extending our previous homoserine linker approach, we prepared a range of conjugates containing one of four different cell or nuclear penetration peptides together with oligonucleotides containing 2'-deoxynucleoside or 2'-O-methylribonucleoside phosphodiesters, or gapmers containing 2'-deoxyphosphorothioates. The route also allows incorporation of a fluorescent label within the conjugate for cell uptake studies.

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2'-aldehydes through thiazolidine, oxime, and hydrazine linkages.

2'-Deoxyoligonucleotides and 2'-O-methyloligoribonucleotides carrying one or more 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy, or hydrazide group to give peptide-oligonucleotide conjugates incorporating single or multiple peptides in good yield. The facile conjugation method allows specific coupling in aqueous solution of unprotected oligonucleotides containing aldehyde groups to unprotected N-terminally modified peptides and other small molecules. A 12-mer 2'-O-methyloligoribonucleotide complementary to the HIV-1 TAR RNA stem-loop and containing two conjugated copies of an 8-mer model laminin peptide was hardly affected in TAR RNA binding and showed a similar level of inhibition of HIV-1 Tat-dependent in vitro transcription compared to the unconjugated 2'-O-methyloligoribonucleotide. Advantages of this conjugation method include (1) the ability to attach more than one peptide or other small molecule to oligonucleotide at defined nucleoside residue locations; (2) a conjugation route that does not affect significantly oligonucleotide binding to RNA structures; and (3) three alternative, facile, and mild conjugation reaction types that do not require use of a large excess of peptide reagent.

Aldehydes↗

PubMedDate not supplied

Use of carbonyl group addition--elimination reactions for synthesis of nucleic acid conjugates.

This review outlines the synthesis of covalent conjugates of oligonucleotides and their analogues that are obtained by reactions of carbonyl compounds with various nucleophiles such as primary amines, N-alkoxyamines, hydrazines, and hydrazides. The products linked by imino, oxime, hydrazone, or thiazolidine groups are shown to be useful intermediates for a wide range of chemical biology applications. Methods for their preparation, isolation, purification, and analysis are highlighted, and the comparative stabilities of the respective linkages are evaluated. The relative merits of incorporation of a carbonyl group, particularly an aldehyde group, into either the oligonucleotide or the ligand parts are considered. Examples of harnessing of aldehyde-nucleophile coupling for the labeling of nucleic acids are given, as well as their conjugation to various biomolecules (e.g. peptides and small molecule ligands), site-specific cross-linking of oligonucleotides to nucleic acid-binding proteins, assembly of multibranched supramolecular structures, and immobilization on functionalized surfaces. Future perspectives of bioconjugation and complex molecular engineering via carbonyl group addition-elimination reactions in nucleic acids chemistry are discussed.

Borohydrides↗

PubMedDate not supplied

1,2-Diol and hydrazide phosphoramidites for solid-phase synthesis and chemoselective ligation of 2'-modified oligonucleotides.

The preparation of two novel 2'-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2'-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO4 to generate 2'-aldehyde oligonucleotides. These were successfully conjugated to acceptor molecules. The latter amidite also showed good coupling yields, but the hydrazide function was demonstrated to be labile under basic deprotection conditions.

Amides↗

PubMedDate not supplied

Total stepwise solid-phase peptide-oligonucleotide conjugate synthesis on macroporous polystyrene.

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2'-deoxynucleoside or 2'-O-methylribonucleoside phosphodiesters, or gapmers containing 2'-deoxyphosphorothioate sequences flanked by 2'-O-methyl wings.

Amino Acid Sequence↗