Sequence specificity of protein-oligonucleotide interactions and protein isolation on sorptive media containing immobilized protein specific oligonucleotides

The sequence specificity of protein—oligonucleotide interactions based on the example of binase interaction with oligodeoxyribonucleotides immobilized in biochip gel elements has been studied. Constants of the preferable binding of binase to the selected nucleotide sequences were measured. The GAGAGAG and GAGAGAGAG oligodeoxyribonucleotides, which specifically bind to binase, were used as molecular probes to develop affine sorptive media for binase isolation and concentration from diluted water solutions. The volume capacity of affinity sorbents with immobilized oligodeoxyribonucleotides was found to be 2.6 and 2.3 mg of binase per 1 mL of sorbent for GAGAGAG and GAGAGAGAG, respectively. It was shown that, after affinity chromatography and elution from sorptive media, the oligonucleotide specificity of binase corresponds to the specificity of the initial sample.     

Biochip development for determining Y-haplogroups that occur in Russian populations

A biochip has been developd that enables one to determine Y-chromosome haplogroups C, DE, G, H, I, J, L, N, O, and R in the DNA sample. SNPs M130, M145, P257, M69, U179, M304, M185, M231, M175, P224 were selected as haplogroup markers, correspondingly. The genotyping included two-round PCR with fluorescent labeling of the product followed by hybridization with immobilized probes on the biochip. An analysis of the ratios of fluorescent signals for immobilized wild-type probe-group specific probe pairs for each of the chosen polymorphic markers showed a high accuracy Y-haplogroup genotyping using the biochip. The reliability of genotyping was confirmed by direct sequencing.     

Geometrical correlations useful for design of sequence-specific DNA narrow groove binding ligands

Isohelical geometry of sequence-specific DNA narrow groove binding ligands was analyzed in terms of H-bond donor/acceptor complementarity between the base pair atoms facing into the narrow groove and the corresponding H-bond donating atoms regularly disposed along the ligand molecule. Spatial correlations found in analytical form were applied to analysis of naturally occurring and hypothetical drug molecule structures. For the case of B-like isohelices the permitted values of the distance L0 between each two neighboring H-bond donating atoms of the ligand as well as the bending angle tau 0 of the line subsequently connecting these atoms were estimated as follows: L0 congruent to (5.0 +/- 0.4) A; tau 0 congruent to (26 +/- 2) degrees.     

Effect of Spacers on DNA Probe Properties in Hybridization Analysis

Russian Journal of Bioorganic Chemistry - A method of immobilizing oligonucleotides on the surface of a polyethylene terephthalate (PET) substrate has been developed. The effectiveness of the...     

Design of Sequence-Specific DNA Binding Ligands That Use a Two-Stranded Peptide Motif for DNA Sequence Recognition

Abstract

The design and DNA binding activity of β-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides - S,S'-bis(Lys-Gly-Val-Cys-Val- NH-NH-Dns) - bridged by an S-S bond binds at least 10 times more strongly to poly(dG)?poly(dC) than to poly(dA)?poly(dT). This peptide can also discriminate between 5′-GpG-3′ and 5′-GpC-3′ steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides - Gly-Cys-Gly- or Val-Cys-Val - bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5′-GpG-3′ steps on DNA, whereas the netropsin-like fragments bind preferentially to tuns of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5′-GpG-3′-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.     

The Use of Thermal Dissociation for Selection of DNA Aptamers

Russian Journal of Bioorganic Chemistry - A method of thermal dissociation of DNA–protein conjugates was developed for the selection of aptamers to human alpha-fetoprotein. The method is...     

Emulsion PCR Amplification of DNA Libraries with Degenerate Central Regions for Aptamer Selection

Russian Journal of Bioorganic Chemistry - For emulsion PCR amplification of oligonucleotide combinatorial libraries the conditions for the stable generation of water-in-oil emulsions with drops of...     

A Simultaneous Use of Cy5-Modified Derivatives of Deoxyuridine and Deoxycytidine in PCR

Russian Journal of Bioorganic Chemistry - Particular features of the simultaneous incorporation into the growing DNA chain of modified Cy5-deoxyuridines (Cy5-dU) and deoxycytidines (Cy5-dC) and Taq...     

Simultaneous Incorporation of Modified dU and dC Derivatives in the Growing DNA Chain Using PEX and PCR

Russian Journal of Bioorganic Chemistry - The substrate efficiency of modified derivatives of 2'-deoxyuridine-(mod-dUTP) and 2'-deoxycytidine-5'-triphosphates (mod-dCTP) was studied in...     

Slippage of the Primer Strand in the Primer Extension Reaction with Modified 2'-Deoxyuridine Triphosphates

Russian Journal of Bioorganic Chemistry - An effect of primer strand slippage was observed in the primer extension reaction due to replacement of thymidine triphosphate for modified analogs of...