[Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of ß-amyloid with DNA].

Interaction of intranuclear ß-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of ß-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of ß-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the ß-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aß facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

[Physico-chemical methods for studing ß-amyloid aggregation].

Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, a key event of the Alzheimer's disease pathogenesis is a transition of the ß-amyloid peptide (Аß) from the monomeric form to the aggregated state. The mechanism of Ðß aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The paper reviews both the well-known and recently introduced physico-chemical methods for analysis of Ðß aggregation, including microscopу, optical and fluorescent methods, method of electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Merits and drawbacks of the methods are discussed. The unique possibility to simultaneously observe Ðß monomers as well oligomers and large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy is emphasized. The high detection sensitivity of the latter method, monitoring the aggregation process in Ðß solutions at low peptide concentrations is underlined. Among mass spectrometric methods, the ion mobility mass spectrometry is marked out as a method enabling to obtain information about both the spectrum of Ðß oligomers and their structure. It is pointed out that the use of several methods giving the complementary data about Ðß aggregates is the best experimental approach to studying the process of b-amyloid peptide aggregation in vitro.

[M31R and R335C polymorphic variants of the IL8RA gene in Russian and Buryat patients with atopic bronchial asthma].

To test the M31R and R335C polymorphisms of the Il8RA gene for association with atopic bronchial asthma (BA), the allele and genotype frequency distributions of the polymorphisms were studied in Russian patients from Moscow and Buryat patients from Ulan-Ude. The study involved two Russian groups, one including 291 DNA samples of patients with atopic BA, and the other, 266 DNA samples of healthy people. The two Buryat groups included 124 and 152 DNA samples from patients with atopic BA and healthy people, respectively. The M31R polymorphism proved to be associated with atopic BA in Russians. Allele Arg and genotype Met/Arg suggested a higher risk of BA (OR= 4.45, P = 0.003 and OR = 4.58, P = 0.003, respectively), while allele Met and genotype Met/Metwere associated with a lower risk (OR = 0.22, P = 0.003 and OR = 0.22, P = 0.003, respectively). The R335C polymorphism was not associated with atopic BA in Russians and was in Buryats. Allele Arg and homozygous genotype Arg/Arg suggested a higher risk of the disease (OR = 3.06, P = 0.030 and OR = 3.20, P = 0.027, respectively), while allele Cys and genotype Arg/Cys suggested a lower risk (OR = 0.33, P = 0.030 and OR = 0.31, P = 0.027, respectively). The results support the role of the IL8RA gene in atopic BA.

[CARD15 and TLR4 genes polymorphisms in atopic bronchial asthma].

In order to investigate whether single nucleotide polymorphisms G(+2722)C and 3020insC in CARD15 gene and Asp299Gly in TLR4 gene contribute to atopic bronchial asthma we performed a comparative analysis of alleles and genotypes frequencies of these polymorphisms in Russian patients from Moscow. DNA samples from 283 patients with atopic bronchial asthma and 227 healthy donors were genotyped. There were associations neither of G(+2722)C and 3020insC in CARD15 gene and Asp299Gly in TLR4 gene with asthma nor of markers of CARD15 gene with asthma severity. Haplotype frequency analysis of CARD15 gene polymorphisms did not reveal significant difference between groups. However, a strong association was found between Asp299Gly and asthma severity. Allele Asp of this marker showed association with mild atopic bronchial asthma and allele Gl--with moderate/severe asthma = 0.47, 95% CI [0.24-0.93] i OR = 2.12, 95% CI [1.08-4.18] respectively).

[Comparative thermodynamic analysis of thrombin interaction with anti-thrombin aptamers and their heterodimeric construct].

Aptamers interacting selectively with the anion-binding exosites 1 and 2 of thrombin were merged into dimeric oligonucleotide constructs with use of a poly-(dT)-linker of 35 nucleotides (nt) long. Complexes of thrombin with the aptamers and their hetero- and homodimeric constructs were measured using the optical biosensor Biacore-3000. K(D) values measured for the hetero- and homodimeric constructs were correspondingly 25-30- and 2-3-fold lower than those for the primary aptamers. Analysis of temperature dependencies of K(D) values within the temperature interval of 10 degrees C-40 degrees C has shown that the values of enthalpy change deltaH upon formation of complexes of thrombin with the aptamers and the hetrodimeric construct are close. The value of the entropy change deltaS upon complex formation of thrombin with the aptamer heterodimeric construct was 1.5-2-fold higher than deltaS values for the complexes with the aptamers. The complex formation and dissociation rates increased with the elevation of temperature from 10 degrees C to 37 degrees C. However, the dissociation rate for the complex of thrombin with the heterodimeric construct was evidently lower that that for the complexes with the aptamers.

[Association of interleukin-13 gene polymorphisms with atopic bronchial asthma].

A comparative analysis of allele and genotype distribution of C(-1055)T and R130Q IL13 gene polymorphisms has been performed in Russian patients from the Moscow region. In the study, 283 DNA specimens of atopic bronchial asthma (BA) patients and 227 DNA specimens of healthy donors were used. No association of these markers with ABA development as well as with total IgE concentration has been found. Haplotype frequency analysis did not reveal significant difference between samples. However, significant association ofC(-1055)Tpolymorphism with the disease severity has been revealed (OR = 2.39, 95% confidence interval 1.44-3.98, p = 0.001). Therefore, C(-1055)T polymorphism was shown to be associated with atopic BA progression.

[Photoaptamer heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinking with a target protein].

Using two DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nt in length) to produce aptamer heterodimeric constructs results into affinity enhancement. The apparent dissociation constant (Kd(app)) measured at the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (Kd(app) = 0.2-0.4 nM) which were approximately 30-fold less than for the complexes with the primary aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer used could bind to the exosite 1. The measured value of Kd(app) for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure to the UV radiation at 308 nm of the equimolar mixtures of thrombin with the photoaptamer construct, the equal yield of the crosslinked complexes was observed at concentrations which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.

Use of oligonucleotides conjugated to gold nanoparticles and streptavidin for amplification of optical biosensor signal during detection of telomeric repeats.

Hybridization of telomeric repeats with a complementary oligonucleotide probe was studied by the surface plasmon resonance method. Conjugation of the probe with streptavidin and gold nanoparticles was shown to amplify the signal at similar concentrations of this probe (by 60 and 300 times, respectively). Nanoparticles can be used for biosensor signal amplification in studying the telomerase activity of malignant cells.

[The use of SNP markers for estimation of individual genetic predisposition to diabetes mellitus types 1 and 2].

Single nucleotide polymorphisms (SNPs) are now considered as the most perspective and convenient markers for research of genetic bases of multifactorial diseases. Fast development of technologies for exact screening of great volume of genetic information, construction of genomic maps of SNP-markers promotes development of innovative diagnostic systems on the basis of significant SNP for an estimation of individual genetic risk of development of various diseases. In this review the basic aspects of genetics of a diabetes of type 1 and 2 and an opportunity of use SNP as markers for an estimation of individual genetic predisposition to the given diseases are considered.

[Synthesis of artifical genome as the basis of synthetic biology].

Recent achievements in the whole-genome sequencing especially viral and bacterial ones, together with the development of methods of bioinformatics and molecular biology, have created preconditions for transition from synthesis of genes to assembly of the whole genomes from chemically synthesized blocks, oligonucleotides. The creation of artificial genomes and artificial cells, will undoubtedly render huge influence on a deepening of knowledge of mechanisms of functioning of living systems at a cellular level, on a way of origin and evolution of life, and also on biotechnology of the future, and will generate preconditions for the further development of synthetic biology and nanobiotechnology.

[Aptamers as perspective affine reagents for clinical proteomics].

Application of proteomic results to scientific and medical practice will depend in many respects on progress of affine microchips technologies that determine the continuous search for inexpensive and robust affine reagents alternative to monoclonal antibodies. Among synthetic mimetics of antibodies, the oligonucleotide aptamers are of the greatest interest as affine reagents due to the possibility to automate their selection and due to the low cost of oligonucleotide synthesis. In the review the problems related to the automation and optimization of aptamer selection and to the selection of photoaptamers capable to formation of photo-induced covalent complexes with protein targets have been considered. The existing approaches to the post-selection modification of the aptamers to increase theirs affinity and selectivity to protein targets are discussed.