Figure 2 - uploaded by Vladimir R Chechetkin
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Dissociation curves for pure duplexes and for their complexes with CroVC normalized with their initial values.
Source publication
Binding specificity of mutant V55C disulfide bonded dimeric lambda-Cro protein (CroVC) to double-stranded DNA (dsDNA) was studied using generic hexamer oligonucleotide microchip. The curves of dissociation of hybridized DNA in the presence and absence of CroVC were converted into the effective discriminant constants to assess the relevant thermodyn...
Contexts in source publication
Context 1
... to technical and experimental failures some of dissociation curves have rather irregular non-monotonous dependence on the temperature and have to be discard- ed from further processing. From 1,024 duplexes formed by hybridization with the mixture 5´-NN(A/G)NN(A/G)NN-3éither in the absence or in the presence of CroVC, we chose 898 samples producing regular dissociation curves in both cases. The criteria of filtering were the reproducibility of curves in the direct and control experiments, the monotonous decrease of observable dissociation curves with the increase in temperature, and the absence of very low initial fluorescence signals (see also Reference 5). The typical examples of experimental fluorescence inten- sities corresponding to the dissociation of pure duplexes and duplexes with the bound CroVC are illustrated in Figure ...
Context 2
... i.e., the dissociation curves in the pres- ence of protein shift to longer times or higher temperature range with respect to the dissociation curves in the absence of protein (cf. Figure 2). In the opposite limit K dp < K sp the situation would be reciprocal (cf., e.g., binding with p50 pro- tein studied in Reference ...
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Citations
... We present an alternative microarray method for quantification of binding affinity of a protein to ssDNA. In contrast to most oligonucleotide microchip techniques based on 2D surface immobilization, we immobilize single-stranded octamer oligonucleotides within 3D hemispherical hydrogel pads (8–12). Immobilization of oligonucleotides within the pads allows us to quantify the affinity of a protein by measuring the complete set of 46 = 4096 dissociation curves for protein–oligonucleotide complexes. ...
... Guanyl-specific bacterial ribonuclease binase produced by Bacillus intermedius (EC 3.1.27.3) was used to develop further this approach. The earlier generation of 3D microarrays proved to be quite efficient for quantitative assessment of sequence specificity of low-molecular-weight compounds, ligands and proteins in their interactions with ssDNA, as well as dsDNA (8–11). Specific motifs found using these oligonucleotide microchips were also confirmed by alternative methods (10). ...
Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to
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were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5′-{N}N1N2N3N4N5N6{N}-3′ consisted of a fixed hexamer motif N1N2N3N4N5N6 in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096
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... Real-time recording of dissociation and measurement of affinities enabled us to deduce consensus-binding sequences for p50 protein and lambda-Cro V55C repressor, which correlated well with previously known specificities [53,54]. ...
Gel-based microarrays (biochips) consisting of nanoliter and sub-nanoliter gel drops on hydrophobic substrate are a versatile technology platform for immobilization of proteins and other biopolymers. Biochips provide a highly hydrophilic environment, which stabilizes immobilized molecules and facilitates their interactions with analytes. The probes are immobilized simultaneously with gel polymerization, evenly distributed throughout individual elements, and are easily accessible because of large pores. Each element is an isolated nanotube. Applications of biochips in the studies of protein interactions with other proteins, nucleic acids, and glycans are described. In particular, biochips are compatible with MALDI-MS. Biochip-based assay of prostate-specific antigen became the first protein microarray approved for clinical use by a national regulatory agency. In this review, 3-D immobilization is compared with mainstream technologies based on surface immobilization.
... The data obtained in such experiments are more complicated to interpret in those cases when the binding region in DNA is substantially longer than the gelimmobilized octanucleotides. Chechetkin et al. suggested new methods of result treatment for these models on an example of binding the covalently crosslinked mutant dimer of the phage λ regulator Cro pro- tein [22]. The authors have developed the method of calculating thermodynamic parameters, which allows one to characterize sequences possessing the highest affinity to the protein, despite the weakening of binding cooperativity owing to a short length of the immobilized oligonucleotides and a relatively high level of background signals. ...
... The authors have developed the method of calculating thermodynamic parameters, which allows one to characterize sequences possessing the highest affinity to the protein, despite the weakening of binding cooperativity owing to a short length of the immobilized oligonucleotides and a relatively high level of background signals. Moreover, it has been shown that a motif with the highest affinity may be revealed by a procedure of " tiling " [22]. A statistical treatment of the data reveals a half-site of binding that corresponds to the data obtained by conventional methods. ...
... Unlike the gel-based microchips, the study of DNA– protein interactions using the molecules immobilized on a two-dimensional surface encounters a number of technical problems. In particular, the glass-immobilized proteins are frequently unstable; in order to immobilize the DNA duplexes containing the binding sites, they have to be placed in the context of " neutral " sequences, which can substantially influence the binding in reality [22]. The most efficient approach to study the DNA–protein interactions with GM is based on the data obtained by biochemical, X-ray crystallographic, and other physical methods, which allow one to estimate a general structure of binding sites: the number, size, and mutual arrangement of individual domains. ...
The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.
... The data obtained in such experiments are more complicated to interpret in those cases when the binding region in DNA is substantially longer than the gelimmobilized octanucleotides. Chechetkin et al. suggested new methods of result treatment for these models on an example of binding the covalently crosslinked mutant dimer of the phage λ regulator Cro protein [22]. The authors have developed the method of calculating thermodynamic parameters, which allows one to characterize sequences possessing the highest affinity to the protein, despite the weakening of binding cooperativity owing to a short length of the immobilized oligonucleotides and a relatively high level of background signals. ...
... The authors have developed the method of calculating thermodynamic parameters, which allows one to characterize sequences possessing the highest affinity to the protein, despite the weakening of binding cooperativity owing to a short length of the immobilized oligonucleotides and a relatively high level of background signals. Moreover, it has been shown that a motif with the highest affinity may be revealed by a procedure of "tiling" [22]. A statistical treatment of the data reveals a half-site of binding that corresponds to the data obtained by conventional methods. ...
... Unlike the gel-based microchips, the study of DNAprotein interactions using the molecules immobilized on a two-dimensional surface encounters a number of technical problems. In particular, the glass-immobilized proteins are frequently unstable; in order to immobilize the DNA duplexes containing the binding sites, they have to be placed in the context of "neutral" sequences, which can substantially influence the binding in reality [22]. The most efficient approach to study the DNA-protein interactions with GM is based on the data obtained by biochemical, X-ray crystallographic, and other physical methods, which allow one to estimate a general structure of binding sites: the number, size, and mutual arrangement of individual domains. ...
The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.
Immobilized oligonucleotides form the critical sensing element of many nucleic acid detection
methodologies. However, pressures on performance and versatility, along with an increasing desire
to expand the scope of targets and assay platforms has driven the integration of special modifications
to enhance stability, functionality and binding characteristics. Separately, aptamers, protein-binding
motifs and catalytic nucleic acids have been retailored for use in immobilized formats to exploit
the sensitivity, signalling and throughput capabilities of these novel assay platforms. Developments
in the field of nanotechnology have also utilized immobilized DNA, but as ascaffold for supramolecular
construction and in the synthesis of molecular bioelectronic components. This review endeavours to
examine the expanding capabilities of immobilized nucleic acids as sensing and structural componentry,
including the modifications required, and the technical advances made to utilize this molecule to
its fullest potential.
