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V V Demidov

Publications and source records attributed to V V Demidov.

31 records · Page 2Linked to original sources
PubMed1995

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

Base Sequence↗
PubMed1995

Radiation and thermal stabilities of adenine nucleotides.

We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

Adenine Nucleotides↗
PubMed1995

Modern aspects of planetary protection and requirements to sterilization of space hardware.

The viewpoint of working group of Russian experts on the problem of planetary protection for future manned and unmanned Mars mission is presented. Recent data of Martian environment and on survival of terrestrial microorganisms in extreme conditions were used for detailed analysis and overview of planetary protection measures in regard to all possible flight situations including accidental landing. The special emphasis on "Mars-94" mission was done. This analysis resulted in revised formulation of spacecraft sterilization requirements and possible measures for their best implementation. New general combined approach to spacecraft sterilization was proposed. It includes penetrating radiation and heat treatment of spacecraft parts and components which is to be carried out before the final assembly of spacecraft and gaseous radiation sterilization of the whole spacecraft during the flight to Mars (or from Mars for return missions).

Containment of Biohazards↗
PubMed1994

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.

Bacterial Proteins↗
PubMed1994

Stability of peptide nucleic acids in human serum and cellular extracts.

The stability of a new type of DNA mimic, peptide nucleic acid (PNA) in human blood serum, Eschericia coli and Micrococcus luteus extracts and nuclear and cytoplasmic extracts from mouse Ehrlich ascites tumor cells was investigated using HPLC analysis. Under conditions that caused complete cleavage of a control peptide, adrenocorticotropic hormone fragment 4-10, no significant degradation of the PNAs, H-T10-LysNH2 and H-TGTACGTCACAACTA-NH2, could be detected. Similarly, PNA H-T5-LysNH2 was found to resist attack by fungal proteinase K or porcine intestinal mucosa peptidase at concentrations exceeding those necessary to completely degrade a control peptide, H-Phe-Trp-Tyr-Cys-Phe-Trp-Tyr-Lys-Phe-Trp-Tyr-Lys-OH, by at least 1000- and 30-fold, respectively. Thus PNA is expected to have sufficient biostability to be used as a drug.

Amino Acid Sequence↗
PubMed1993

High-performance liquid chromatography of the photoproducts of nucleic acid components. III. Detection of the secondary structure differences in sequence isomeric self-complementary oligonucleotides.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect differences in the secondary structures of two self-complementary oligodeoxyribonucleotides. The [d(CCTTTAAAGG)]2 duplex assumes an ordinary B-conformation in aqueous solution, while [d(GGAAATTTCC)]2 is known to contain in its central part a stretch of a more rigid B'-form conformation with significantly lowered fluctuational mobility of base pairs. The latter factor causes a marked difference in the amounts of thymine cyclobutane photodimers formed under UV irradiation of corresponding duplexes as revealed by chromatography of two single-stranded oligonucleotides. Increasing the temperature below the duplex melting temperature (stimulation of the B'-B structural transition) results in an increase in photodimer formation that was inhibited in the B'-form. Thus, we demonstrate the usefulness of RP-HPLC for duplex DNA structural studies.

Base Sequence↗
PubMed1993

HPLC photofingerprinting of conformational peculiarities and transitions in oligonucleotide duplexes.

Two self-complementary sequence-isomeric decadeoxyribonucleotides were exposed to UV light under conditions in which they assume duplex structures. After that they were analyzed in the denatured state by reversed-phase high-performance liquid chromatography (HPLC). Characterization of the separated photoproducts allowed localization of cyclobutane pyrimidine dimers in the sequences of the modified oligonucleotides. For [d(GGAAATTTCC)]2, which is known to contain in its central part a stretch of rigid B'-conformation with decreased mobility of constituent bases, lower yields of thymine dimers, as compared with that for ordinary B-form [d(CCTTTAAAGG)]2, were found. On the contrary, mixed thymine-cytosine heterodimers generated in the former oligonucleotide demonstrate the increase in photoreactivity of these residues at the B'-B junction. This is probably due to the peculiar conformation adopted by this decanucleotide. Stimulation of B'-B transition, by increasing the temperature before melting, reduced an inhibition of thymine photodimer formation. During the melting of both oligonucleotides yields of all identified photoinduced cyclobutadipyrimidines were reduced. Possible influences of some metal cations on the stability of the B'-form were also studied by this photoprobing technique. The present study demonstrates the feasibility of HPLC photofingerprinting as a new approach for structural analysis of nucleic acids.

Base Sequence↗
PubMedDate not supplied

[Kinetics of formaldehyde splitting off from the hydroxymethylated amino groups of nitrogen bases incorporated into double-helical DNA].

We studied the kinetics of formaldehyde dissociation from hydroxymethylated amino groups of nitrous bases on native DNA. Compared with monomers, the rate constant of formaldehyde dissociation from such bases integrate in the double helix proved to be 20 times smaller for adenine and 4 times smaller for cytidine within the temperature range of 15 to 40 degrees C. The kinetic pattern suggests that the dissociation of formaldehyde from hydroxymethylated amino groups does not occur in the direction of the base plane nor through a full fluctuational opening of base pairs. It is presumed that formaldehyde dissociation from modified amino groups is due to softer fluctuational changes which however, make it possible for formaldehyde to attack amino groups perpendicularly to the base plane.

Chemical Phenomena↗