T. S. Oretskaya

Conjugates of Oligonucleotides and Analogues with Cell Penetrating Peptides as Gene Silencing Agents

The review describes key aspects of the synthesis and biological activities of conjugates of oligonucleotides and their analogues with synthetic peptides, in particular aimed towards gene silencing applications. The common methods of synthesis of oligonucleotide-peptide conjugates (OPCs) and PNA-peptide conjugates (PPCs) are described, which include both total solid-phase and fragment coupling approaches. In addition, various applications of conjugates as gene silencing agents are outlined. These include antisense and steric block applications in mammalian cells of OPCs, PPCs and phosphorodiamidate morpholinooligonucleotide (PMO)-peptide conjugates, gene silencing in bacteria, various DNA targeting applications, and recent reports of gene silencing activities of siRNA-peptide conjugates. A table listing all peptides used as oligonucleotide conjugates for gene silencing applications is also included.

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA.

The interaction of nucleotide excision repair factors-xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)—with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.

The behaviour of 2'-deoxy-2'-fluorouridine incorporated into oligonucleotides by the phosphoramidite approach

Abstract 2′-Deoxy-2′-fluorouridine has been chemically incorporated into an oligodeoxynucleotide of the structure 5′ACGGAX 3′ (X=U(2′-F)) using the phosphoramidite method and the behaviour of the product has been studied. 5′-O-Monomethoxytrityl-2′-deoxy-2′-fluorouridine was fixed on silica gel at the 3′-end and the chain elongated on a DNA-synthesizer using nucleoside methoxyphosphoramidites. After alkaline work-up two products were observed. One was found to be the desired fluoro containing hexamer, whereas the other corresponds to an araU-hexamer (X=arabino-furanosyluridine). The latter compound is supposed to be a product of alkaline hydrolysis of the C-2′-F-bond. The oligomers containing 2′-fluoro- and ara-U at their 3′-end were chemically sequenced by a solid phase method on CCS-paper which confirmed the right primary structure.

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments: EcoRII endonuclease cleavage of substrates with repeated natural and modified recognition sites

Interaction of EcoRII restriction endonuclease with a set of synthetic concatemer DNA duplexes with natural and modified sites for this enzyme has been studied. DNA duplexes with repeated natural sites are cleaved by EcoRII. Substitution of central AT‐pair in the recognition site for a non‐complementary TT‐ or AA‐pair reduces the rate of cleavage, this effect being much more pronounced in the last case. Absence of site flanking in one strand from the 5′‐terminus also results in very slow cleavage. The results obtained testify to the interaction of EcoRII with both strands of the substrate.

Cyanogen Bromide-Induced Chemical Ligation: Mechanism and Optimization of the Reaction Conditions

Abstract Chemical ligation induced by BrCN in the presence of N-substituted morpholine has been studied in details. It has also been shown that the phosphate activation by BrCN can be used for the synthesis of nucleotide derivatives in aqueous solutions.

Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H.

Chimeric oligo(ribo‐deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11.

CHEMICAL CROSS-LINKING OF MVAI AND ECORII ENZYMES TO DNA DUPLEXES CONTAINING MONOSUBSTITUTED PYROPHOSPHATE INTERNUCLEOTIDE BOND

DNA duplexes containing a monosubstituted pyrophosphate internucleotide group, instead of a phosphodiester bond, were used as cross-linking reagent for the affinity modification of the restriction endonucleases EcoRII and MvaI (R.EcoRII and R.MvaI). An active group was introduced into the enzymes recognition site or between the recognition site and flanking sequence. The substrate properties of such DNA duplexes were determined. Cross-linking specificity was demonstrated by competition experiments with unmodified substrate, as well as by the absence of cross-linking to an active duplex lacking a recognition site. It was shown that the nucleophilicity of the buffer solution and the presence of the enzyme cofactor Mg2+ dramatically affected the cross-linking yield.

Affinity modification of the restriction endonuclease SsoII by 2'-aldehyde-containing double stranded DNAs.

Properties of 2′-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.

Synthesis of 2′-Modified Oligonucleotides Containing Aldehyde or Ethylenediamine Groups

Abstract Oligonucleotides carrying 2′-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2′-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2′-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.

Synthesis of Modified Oligonucleotides and Production of Duplexes With Covalently Linked Chains

Abstract A method has been devised to synthesize a DNA-duplex with covalently connected strands. Primary amino group located on one strand is linked to a carboxyl group of the other strand through the agency of a water soluble carbodiimide condensing agent. Conditions for the reaction between chains of a duplex composed of the modified oligonucleotides [1] were optimized. The thermal and hydrolytic stability of the cross linked duplex was examined.