Biomedical subjects

V N Tashlitsky

Publications and source records attributed to V N Tashlitsky.

5 recordsLinked to original sources

PubMed1999

Oligonucleotide-peptide conjugates as potential antisense agents.

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.

Adenosine↗

PubMed1998

EcoRII endonuclease has two identical DNA-binding sites and cleaves one of two co-ordinated recognition sites in one catalytic event.

EcoRII is a typical restriction enzyme that cleaves DNA using a two-site mechanism. EcoRII endonuclease is unable to cleave DNA which contains a small number of EcoRII recognition sites but the enzyme activity can be stimulated in the presence of DNA with a high frequency of EcoRII sites. To investigate the mechanism of activation, the kinetics of stimulated EcoRII cleavage has been studied. A 14 bp substrate activated the cleavage of the 71 bp substrate, containing one EcoRII recognition site (trans-activation) by a competitive mechanism: the activator increased substrate binding but not catalysis. The activation increased if the substrate concentration decreased and if the activator had a lower affinity for the enzyme than the substrate. The introduction of the second recognition site into the 71 bp duplex also enabled cleavage of this substrate (cis-activation). Pyrophosphate bonds were incorporated into one of two recognition sites to switch off the cleavage of the phosphodiester bonds. Analysis of cleavage products of these modified substrates showed that EcoRII cuts one of two coordinated recognition sites in one catalytic event.

Base Sequence↗

PubMed1996

Synthesis and properties of cross-linked DNA duplexes.

A method has been devised to synthesize DNA duplexes with covalently connected strands. The structure of cross-linked duplexes was confirmed by a reaction with the restriction endonuclease AluI. The thermal stability of the resulting compounds was investigated.

Base Sequence↗

PubMed1995

Use of UV spectroscopy for the study of nucleic acid cleavage by E. coli RNase H and restriction endonucleases.

A one-step spectrophotometric method for monitoring of nucleic acid cleavage by ribonuclease H from E. coli and type II restriction endonucleases has been proposed. It is based on recording of the increase in the UV absorbance at 260 nm during the course of enzymatic reaction. Duplexes stable under the reaction conditions were chosen as substrates for the enzymes being studied. In order to obtain duplex dissociation following their cleavage by the enzyme appreciate temperature conditions were selected. The spectrophotometric method may be applied for rapid testing of the nuclease activity in protein preparations as well as for precise quantitative analysis of nucleic acid degradation by enzymes. This method may be successfully employed in kinetic studies of nucleic acid-protein interactions.

Base Sequence↗

PubMed1992

Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII.

A study was made of the interaction between restriction endonucleases recognizing CCNGG (SsoII and ScrFI) or CCA/TGG (MvaI and EcoRII) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy] methylguanine (gIG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for gIG) introduced into the recognition site both increase the efficiency of SsoII and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by SsoII occurs at the phosphodiester bond adjacent to the point of modification towards the 5'-end. With the guanine base returned (the substrate with gIG), the correct cleavage position is restored. ScrFI specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the gIG-containing duplex) are resistant to hydrolysis by MvaI and EcoRII. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that SsoII forms a complex with DNA in an 'open' form.

Base Sequence↗