Bionanoconjugate for detection and isolation of nucleic acids and method of its production

FIELD: biotechnology.

SUBSTANCE: bionanoconjugate comprises a nano-sized superparamagnetic particle of cobalt ferrite spinel CoxFe3-xO4, where 0.6≤x≤0.98, obtained by mechanochemical synthesis. To isolate the nucleic acid containing oligo- or poly-A/dA sequence the synthetic single stranded oligonucleotide 5'-dGndTm is used, where n=5-30, m=10-35, one portion of which, consisting of guanine nucleotides in the presence of phosphate anions in the solution is specifically associated with the surface of the nanoparticle and the other - consisting of thymine nucleotides is able to enter into hybridisation with oligo- or poly-A/dA nucleotide sequences. For isolation from the solution in the presence of phosphate anions of specific hetero-nucleotide sequence additionally create a molecule of oligonucleotide-adapter containing the sequence of oligo-dAx at the 3'-end, hybridised with thymine nucleotides of a complex 5'-dGndTm, where n=5-30, m=10-35, and a portion at the 5'-end complementary to a specific hetero-nucleotide sequence in the solution.

EFFECT: invention enables to detect effectively and to isolate the single-stranded nucleic acids.

3 cl, 8 dwg, 1 tbl, 5 ex

 

The invention relates to medicine, namely to create conjugates magnetic particle - nucleic acid required for molecular genetic diagnosis.

The creation of new efficient systems and bionanomatrix designs for the diagnosis of various diseases and other problems is an important area of modern medicine and biology. One of the main objectives is the creation of conjugates, in particular, for subsequent detection and selection of various organs and tissues of the genetic material (DNA, RNA), on the basis of research which will be diagnosed. Methods of detection and selection of nucleic acids should have a high sensitivity and specificity, to be simple and fast, to give reproducible results and to ensure high purity of the DNA/RNA.

The most promising discovery and selection of nucleic acids is a method of magnetic separation, in which as sorbents are magnetic nanoparticles, mixed in solution with biological structures and forming with the latter conjugates. In most cases, these particles consist of iron oxides, the surface of which is coated with various substances and functional groups, contributing to the formation of conjugates. When on the ogenyi magnetic field with a sufficiently large gradient of the magnetic particles, creating conjugates with nucleic acids (DNA/RNA), can be separated from the solution and subjected to further processing.

The main advantages of using magnetic nanoparticles for detection and selection of nucleic acids due to the following:

magnetic nanoparticles are single, have a super-paramagnetic behavior and magnetic properties required for the selection of biological objects using external magnetic fields.

The present invention is the creation of bionanocomposite with a high degree of selectivity for separation of single-stranded nucleic acids from the solution.

The problem is solved by the fact that as the basis bionanocomposite take nanosized superparamagnetic particles particle cobalt ferrospinel CoxFe3-xO4where 0.6<x≤0.98, obtained by the method of mechanochemical synthesis (EN 2471502, 2013), and to isolate nucleic acids containing oligo - or poly-A/dA sequence, use a synthetic single-stranded oligonucleotide 5'-dGndTmwhere n=5 to 30, m=10-35, one part of which, consisting of Gurinovich nucleotides, in the presence of phosphate anions in solution specifically associated with the surface of the nanoparticles, and the other consisting of siminovich nucleotides capable to enter into GI is ralizatio with oligo - or poly-A/dA nucleotide sequences and for separation from a solution containing phosphate anions, specific heteronuclei sequences use the molecule of the oligonucleotide adapter containing the sequence of the oligo-dAxon the 3'-end, hybridizing with terminowymi nucleotides of the 5'-dGndTmwhere n=5 to 30, m=10-35, and a site at the 5'end, complementary to the specific nucleic acid sequence in solution.

When n<5 linking the nanoparticles with the nucleic acid is weak. When n>30 percentage of allocated product almost does not change and, thus, further increasing the number Gurinovich nucleotides does not lead to significant advantages. Accordingly, when m<10 relationship between d-"tail" bionanocomposite and the complementary nucleic acid is unstable under normal conditions (instability formed through hydrogen bonds a-T double-stranded hybrid at room temperature), and if m>35 difficulties with subsequent separation allocated nucleic acid from bionanocomposite (high melting point And T-bonds).

Compared to other technical solutions proposed bionanocomposite has advantages, which are as follows:

a) To create bionanocomposites use nanoparticles of cobalt ferrospinel Cox Fe3-xO4where 0.6≤x≤0.98 with an average diameter in the range of 3-11 nm, obtained by the method of mechanochemical synthesis and possessing super-paramagnetic behavior.

Unlike other methods of chemical condensation during mechanochemical synthesis of the impact of intense deformation (impact, friction) leads to the formation of nanoparticles in the "active" non-equilibrium States, which are characterized by high elastic microstresses (∆D/d=(5.4-7.4)·10-3), violations of the degree of order in the arrangement of heterogeneous ions, the change in the lattice parameters, the amorphization of the surface layer. The crystal lattice of such nanoparticles is large displacements of the ions, the chemical composition is essentially non stoichiometry, therefore, obtained by mechanochemical synthesis of nanoparticles have a large stored energy and are metastable and "active". It can be assumed that the presence of "active" nonequilibrium States facilitates the formation of conjugates between DNA and metal cations on the surface of the nanoparticles.

Installed, for example, that the efficiency of DNA transfer into cells in vitro by magneticly nanoparticles synthesized by mechanochemical method, noticeably superior to the same size of the nanoparticles obtained by chemical vapor deposition (M. A. Sukoyan, E. A. Khrapov, E. N. Voronina et.al. Magnitofection of Human Smatic Cells with Magnetite and Cobalt Ferrospinel Nanoparticles/Bulletin in Experimental Biology and Medicine. 2013, v.154, No. 5, March, pp. 673-676).

b) Magnetic nanoparticle cobalt ferrospinel does not require coating, widely used in other technical solutions, as proposed chemically synthesized oligonucleotide carries out direct linking directly with the surface of the nanoparticles at the expense of plot, represented by the repetition of Gurinovich nucleotides. The use of magnetic nanoparticles uncoated leads to an increase of the magnetic properties, which are usually reduced in the coating increases the surface area for binding with biomolecules and allows you to more easily create a colloidal system. At the same time as a result of this functionalization is achieved by blocking the adsorption-active centers in relation to the nucleic acids on the surface of the nanoparticles.

It is established that in the case of nanoparticles uncoated extraction procedure is simple, fast, cheap and reliable, does not require organic solvents and can be easily automated (Saiyed Z. M., Ramchand. Extraction of Genomic DNA Using Magnetic Nanoparticles (Fe3O4) as a Solid-Phase Support/Americal Journal of Infectious Diseases 3(4): 225-229, 2007; Saiyed Z. M., C. N. Ramchand, Telang S. D. Isolation of genomic DNA using magnetic nanoparticles as a solid phase support/J. Phys.: Condens. Matter, v.20 (2008) 204153).

in the Proposed bionanocomposite provides a high degree of selectivity close to 100% when selected and oligo - or poly A/dA nucleotide sequences from the solution, containing phosphate anions at a concentration of (10±0.5)·10-3mol/L. After joining molecules"adapter" bioconjugate provides the same degree of selectivity in the allocation of specific heteronuclear sequences. An important role is played phosphate anions, which are in the selected concentration to prevent nonspecific binding.

The method of obtaining bionanocomposite for separation of single-stranded nucleic acids, which consists in the fact that for the selection of nucleic acid containing oligo - or poly-A/dA sequence, take the super-paramagnetic nanoparticles cobalt ferrospinel CoxFe3-xO4where 0.6≤x≤0.98 obtained by mechanochemical synthesis, the nanoparticles were washed with distilled water to prepare aqueous suspension of the washed nanoparticles, the suspension is separated into fractions in the form of sediment and nadeshiko selected adosados and mix with it a synthetic single-stranded oligonucleotide 5'-dGndTmwhere n=5 to 30, m=10-35, and sodium phosphate at a concentration of (10±0.5)·10-3mol/l, after which the mixture is incubated before the establishment of full adsorption equilibrium and produce bionanocomposite method of magnetic separation, followed by centrifugation and washing, and to highlight heteronuclei sequence in a solution containing nano is astitsy cobalt ferrospinel, associated with synthetic single-stranded-oligonucleotide 5'-dGndTmwhere n=5 to 30, m=10-35, and sodium phosphate at a concentration of (10±0.5)·10-3mol/l, add molecules of the oligonucleotide-"adapter" that contains the sequence of the oligo-dAxon the 3'-end and a site at the 5'end, complementary to the specific heteronuclei sequence in solution, after which the mixture is again incubated before the establishment of full adsorption equilibrium and produce bionanocomposite method of magnetic separation, followed by centrifugation and washing. In this case n preferably equals = 20-25, a m is preferably 15-25.

The concentration of the border of the content of phosphate in the buffer solution is determined by the following considerations. When the content of sodium phosphate less than (10±0.5)·10-3mol/l plot of the oligonucleotide represented terminowymi nucleotides, can enter into binding with the surface of the nanoparticles. Increasing the concentration of phosphate in the solution above (10±0.5)·10-3mol/l can lead to a decrease in the density of coating the surface of nanoparticles.

Example 1. Getting bionanocomposite.

For the preparation of suspensions make a super-paramagnetic nanoparticles cobalt ferrospinel Co0.9Fe2.1O4obtained by mechanochemical synthesis, in 10 mm Tris (pH 5.0) to the end to which ncentratio 0.2 mg/ml Treat a suspension of 20 minutes ultrasound at a frequency of 22 kHz, a power of 25 watts (Bandelin Sonopuls HD 2070) and centrifuged at 13400 rpm for minutes (Eppendorf MiniSpin) for the deposition of aggregates of nanoparticles. Select the supernatant portion representing a suspension of nanoparticles of cobalt ferrospinel with a concentration of 0.15 mg/ml (pH 6.5).

To obtain bionanocomposite to 8/10 parts of the resulting suspension of the nanoparticles add 1/10 volume of an aqueous solution of the corresponding oligonucleotide composition dG18dT25with a concentration of 50*10-6mol/l and 1/10 volume of 10x phosphate-saline buffer (PBS) (10xPBS:1.37 M NaCl, 0.027 M KCl, 0.1 M Na2HPO4, 0.02 Μ ΚΗ2ΡO4, pH 7.4) and stirred on the vortex (BioSan FV-2400). Next incubated the reaction mixture under normal conditions (ambient temperature 20±10°C, relative humidity from 40 to 60%, atmospheric pressure of 760±20 mm RT.CT.) within 24 hours to determine full adsorption equilibrium. Received binalatongan separated from the solution by the method of magnetic separation on a magnetic stand (Promega), centrifuged for 15 minutes at 13400 rpm and washed twice with bidistilled water and Tris-containing buffer.

Figure 1 shows the IR spectra of nanoparticles of cobalt ferrite (Co0.9Fe1.1O4after incubation in phosphate-containing buffer, ol is gonucleotide (dG 18T25and bionanocomposite (dG18T25-Co0.9Fe1.1O4), obtained according to example 1.

The IR spectrum was obtained in the D2O and H2O by the method of the ATR diamond crystal (Nicolet 6700, Thermo). The presence in the IR spectrum bionanocomposite characteristic absorption bands: cobalt ferrite at 588 cm-1caused by vibrations of the metal-oxygen in tetrahedral oxygen interstitials ferrite, and the oligonucleotide at 1083 cm-1and 1210 cm-1due to symmetric and asymmetric vibrations of phosphate, and in the field of 1500-1700 cm-1fluctuations of groups of bases, confirms the formation bionanocomposite. The observed change in the position of absorption bands in the IR spectrum bionanocomposites compared with unrelated oligonucleotide (table 1) is a consequence of the interaction of groups of the sugar-phosphate backbone and bases of guanine with charged atoms on the surface of the nanoparticles.

Figure 2 shows the efficiency of binding of the oligonucleotide dG18T25with nanoparticles of different composition. It is seen that the nanoparticles containing cobalt in the range 0.69-0.90 effectively bind the oligonucleotide of the solution (Inmax=2.7±0.2, Kd=0.36±0.07·10-6M) and form nanobioconjugates.

The specific magnetization of the ensemble bonnacon Ulatov (MAGNETOMETER N-04) is 20±0.4 Gauss·cm 3/, a Slight decrease of the magnetic properties compared with the original ensemble of nanoparticles of cobalt ferrite (22 Gauss·cm3/g) is a consequence of the reduction of interparticle interactions in an ensemble of nanoparticles because of the increased distance between them.

Example 2. Getting bionanocomposite for separation of nucleic acid containing oligo(dA) sequence at the 3'-end.

Bionanocomposite obtained according to example 1, after cleaning with water, washed with buffer, 0.5 M NaCl, 10 mm Tris (7.5-8.0) and diluted in the same buffer to a concentration of 5 mg/ml In vitro-type Eppendorf mix 20 μl of the suspension containing binalatongan, 60 μl of buffer 0.5 M NaCl, 10 mm Tris (7.5-8.0), 10 μl of 10x phosphate-saline buffer (PBS) (10xPBS: 1.37 M NaCl, 0.027 M KCl, 0.1 M Na2HPO4, 0.02 Μ ΚΗ2ΡΟ4, pH 7.4) and 10 μl of an aqueous solution of the oligonucleotide dΑ2525 (5'-aaaaaaaaaaaaaaaaaaaaaaaaaa-3'). Stir the mixture in the vortex (BioSan FV-2400). Stand 10 minutes under normal conditions and again shaken on a vortex. Precipitated on the magnetic stand (Promega) for 10 minutes. The supernatant is removed. Double-washed precipitate buffer 0.8 Μ NaCl, 10 mm Tris (7.5-8.0). Add 20 ál of water.

Figure 3 shows the efficiency of binding (nmol/mg) synthetic oligonucleotide containing 25 adenine residues (dA25from a solution of 537 mm NaCl, 8 mm Tris, 2.7 mm KCl, 10 mm Na 2HPO4, 2 mm KN2RHO4(pH 7.5), at varying concentrations of dA25from 0.25 to 5 µmol/l using bionanocomposite. The effectiveness of subsequent elution in water at a temperature of 70°C was 96,0±0,8%.

Figure 4 shows the efficiency of binding (nmol/mg) synthetic oligonucleotide containing oligo(dA) sequence at the 3'-end dN23dA25(5'-tctggtaaagtggatattgttgcaaaaaaaaaaaaaaaaaaaaaaaaa-3') using bionanocomposites obtained based on nanoparticles of different composition.

Example 3. The allocation of the poly+mRNA using nanobioconjugates.

After cleaning with water bionanocomposite washed with buffer with 0.5 M NaCl, 10 mm Tris (7.5-8.0) and diluted in the same buffer to a concentration of 5 mg/ml In vitro-type Eppendorf mix 10 μl of the suspension containing binalatongan, 70 μl of buffer with 0.5 M NaCl, 10 mm Tris (7.5-8.0), 10 μl of 10x phosphate-saline buffer (PBS) (10xPBS: 1,37 Μ NaCl, 0,027 Μ KCl, 0,1 Μ Na2HPO4, of 0.02 Μ ΚΗ2ΡO4, pH 7.4) and 10 μl of the investigated samples (total RNA) preparation of total RNA pre-heated at 65°C for 5 minutes). Carefully mix the mixture. Stand 5 minutes under normal conditions and again mix. Precipitated on the magnetic stand (Promega) for 5 minutes. The supernatant is removed. Double-washed precipitate buffer with 0.5 M NaCl, 10 mm Tris (7.5-8.0). Add 20 ál of ionizovannoi water (free from RNase), mix thoroughly and incubate at 70°C for 10 minutes in a solid state thermostat (Termite, DNA-technology). Precipitated bionanocomposite magnetic tripod and select the supernatant containing the poly+mRNA, into a clean tube. Preparation of mRNA can be used for further molecular genetic manipulations.

Preparation of total RNA from mononuclear cells venous blood was obtained using TRIzol-reagent (Life technologies) according to the instructions of the manufacturer. Allocated using bionanocomposite mRNA from the total RNA preparation was used for cDNA synthesis using the set for the given reaction reverse transcription Mint-revertase (Evrogen). Synthesized cDNA was used for PCR in real time using sets for PCR-RV in the presence of SybrGreen I (Synthol) using primers for mRNA/cDNA gene glitserofosfatdegidrogenazy. Figure 5 shows amplification curves obtained by PCR-RV (LightCycler 480, Roche). For the given reaction took cDNA synthesized on the matrix mRNA allocated by using bionanocomposite (curve a) and total RNA (curve b). Curves C, d, e, f - series serial tenfold dilutions of control cDNA(1:10, 1:100, 1:1000, 1:10000).

Thus, the implemented technical solution - bi is nanoconjugate - ensures effective (>1.2 µmol/g) binding to molecules of the specific nucleic acid having a specific sequence of nucleotides in solution, which can be separated (bweremana) from bionanocomposite with an efficiency of at least 95%. The elution efficiency far exceeds the maximum value of this indicator for the prototype (58%). Low efficiency of elution can be a source of false-negative results (undiagnosed) for use in molecular genetic diagnostics. The ability bionanocomposite unlike the prototype to bind to specific molecules of DNA/RNA can be used for detection of diagnostically important nucleic acids in the sample, for example with fluorescent or magnetic resonance detection.

Example 4. Getting bioconjugate to highlight specific nucleic acid (specified composition) of the mixture.

Bionanocomposite obtained according to example 1, washed in buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HPO4, 0,002 Μ KH2PO4, pH 7.4) and diluted in the same buffer to a concentration of 5 mg/ml In vitro-type Eppendorf mix 50 μl of the suspension containing binalatongan, 50 μl of the oligonucleotide-"adapter" dN23dA2525·10-6Μ (5'-gcaacaatatccactttaccagaaaaaaaaaaaaaaaaaaaaaaaaaa-3') and bring the volume buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HPO4, 0,002 Μ KH2PO4, pH 7.4), 500 ál. Stir the mixture in the vortex (BioSan FV-2400). Stand 15 minutes under normal conditions and again shaken on a vortex. Precipitated bioconjugate magnetic separation and centrifugation. The supernatant is removed. Double-washed precipitate buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HPO4, 0,002 Μ KN2RHO4, pH 7.4). Dissolved in the same buffer in a final volume of 50 µl.

10 µl of the obtained bionanocomposite, hybridizing molecules of the oligonucleotide-"adapter" dN23dA25are mixed in a test tube type Eppendorf with 90 µl of a mixture containing two types of oligonucleotides in a concentration of from 0.5 to 0.7·10-6M labeled with different fluorescent labels.

The mixture is incubated at 37°C for 20 minutes. Precipitated bioconjugate magnetic separation and centrifugation. Adosados selected, the precipitate washed twice with buffer: 10 mm Tris, 0,137 Μ NaCl, 0.0027 M KCl, 0.01 To Μ Na2HPO4, 0.002 Μ KH2PO4, pH 7.4. Sediment resuspended in water and heated for 15 minutes at 70°C for elution of the oligonucleotide from bionanocomposite.

Figure 6 shows the efficiency of the binding (hybridization) of synthetic oligonucleotides Cy3-dN23(5'-tctggtaaagtggatattgt-/Cy3/-3') and Cy5-dN19(5'-/SW/-aatggtgtctgagcgatgtg-3') of a mixture of buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HO 4, 0,002 Μ KH2PO4, pH 7.4) with a variable portion of the molecule is"adapter" dN23dA25(5'-gcaacaatatccactttaccagaaaaaaaaaaaaaaaaaaaaaaaaaa-3'), which is part of bionanocomposite.

You can see that the efficiency of binding of the complementary variable region of a molecule"adapter" of the oligonucleotide Cy3-dN23reaches 99%, whereas complementary oligonucleotide Cy5-dN19not associated with bioconjugates under these conditions.

Figure 7 shows the results of electrophoretic analysis 1.2% agarose gel aqueous solutions of oligonucleotides, selected using bionanocomposite (elution) (tracks 1-3), supernatant after incubation of the mixture of oligonucleotides with bionanocomposites (tracks 4-6), source samples (lanes 7-9) containing a mixture of complementary and complementario oligonucleotides (control) at a concentration of from 0.5 to 0.7·10-6M Can be observed that Cu-labeled oligonucleotide, complementary variable part of the oligonucleotide-"adapter", remains in the supernatant, whereas the complementary SS3-labeled oligonucleotide binds to bioconjugates and can be subsequently separated from bioconjugate (blueraven) is water.

Example 5. Getting bionanocomposite for detection and selection of specific nucleic acid (oligonucleotide a given composition).

Bananarang is t, obtained according to example 1, washed in buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HPO4, 0,002 Μ KH2PO4, pH 7.4) and diluted in the same buffer to a concentration of 5 mg/ml In vitro-type Eppendorf mix 50 μl of the suspension containing binalatongan, 50 μl of the oligonucleotide-"adapter" dN21dA25at a concentration of 25·10-6Μ (5'-aagcttatcagactgatgttgaaaaaaaaaaaaaaaaaaaaaaaaa) and bring the volume of buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HPO4, 0,002 Μ KH2PO4, pH 7.4), 500 ál. Stir the mixture in the vortex (BioSan FV-2400). Stand 15 minutes under normal conditions and again shaken on a vortex. Precipitated bioconjugate magnetic separation and centrifugation. The supernatant is removed. Double-washed precipitate buffer (10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ Cl, 0,01 Μ Na2HPO4, 0,002 Μ KH2PO4, pH 7.4). Dissolved in the same buffer in a final volume of 50 µl.

10 µl of the obtained bionanocomposite, hybridizing molecules of the oligonucleotide adaptors dN21dA25(5'-aagcttatcagactgatgttgaaaaaaaaaaaaaaaaaaaaaaaaa-3'), are mixed in a test tube type Eppendorf with 90 μl of a mixture containing selected SS3-dN21(5'-caacatcagtctgataagct-/Cy3/-3'), Cy3-dN23(5'-tctggtaaagtggatattgt-/Cy3/-3') oligonucleotide in a concentration of from 0.1 to 0.8·10-6M labeled fluorescein label (SS3). The mixture is incubated at 37°C for 20 minutes. Precipitated bionanocomposite magnetic behold what aracia and centrifugation. Adosados selected, the precipitate washed twice with buffer: 10 mm Tris, 0,137 Μ NaCl, 0,0027 Μ KCl, 0,01 Μ Na2HPO4, 0,002 Μ KH2PO4, pH 7.4. Sediment resuspended in water and heated for 15 minutes at 70°C for elution of the oligonucleotide from bionanocomposite.

Figure 8 shows the results of electrophoretic separation of samples obtained by elution, and supernatants obtained after keeping bioconjugate with complementary (lane 1 and 1s) and complementary (in a concentration of from 0.1 to 0.7·10-6M) the variable portion of the molecule- ' adapter oligonucleotide (lane 2-8 and 2s-8s). You can see that during incubation with acomplementary the oligonucleotide, it is not associated with bioconjugates (lane 1) and remains in the supernatant (lane 1), whereas the complementary variable region of a molecule"adapter" oligonucleotide effectively communicates from the solution (tracks 2s-8s) and can be blueraven later from bioconjugate (tracks 2-8). The effectiveness of subsequent elution in water at temperatures up to 70°C amounted to 96.0±0.8%.

1. Bionanocomposite for separation of single-stranded nucleic acid that represents a specific heteronuclear nucleic acid or nucleic acid-containing oligo - or poly-A/dA sequence, including nanoscale super is arabinitol particle cobalt ferrospinel Co xFe3-xO4where 0.6≤x≤0.98 obtained by mechanochemical synthesis, for the selection of nucleic acid containing oligo - or poly-A/dA sequence, use a synthetic single-stranded oligonucleotide 5'-dGndTmwhere n=5 to 30, m=10-35, one part of which, consisting of Gurinovich nucleotides, in the presence of phosphate anions in solution specifically associated with the surface of the nanoparticles, and the other consisting of siminovich nucleotides capable to enter into the hybridization with oligo - or poly-A/dA nucleotide sequences, and for separation from solution in the presence of phosphate anions specific heteronuclei sequence optionally use the molecule of the oligonucleotide adapter containing the sequence of the oligo-dAxon the 3'-end, hybridizing with terminowymi nucleotides of the complex 5'-dGndTmwhere n=5 to 30, m=10-35, and a site at the 5'end, complementary to the specific heteronuclei sequence in solution.

2. The method of obtaining bionanocomposite for separation of single-stranded nucleic acids under item 1, namely, that for the selection of nucleic acid containing oligo - or poly-A/dA sequence, take the super-paramagnetic nanoparticles cobalt ferrospinel CoxFe3-xO4where 0.6≤x≤0.98, polucen the e by mechanochemical synthesis, the nanoparticles were washed with distilled water to prepare aqueous suspension of the washed nanoparticles, the suspension is separated into fractions in the form of sediment and nadeshiko selected adosados and mix with it a synthetic single-stranded oligonucleotide 5'-dGndTmwhere n=5 to 30, m=10-35, and sodium phosphate at a concentration of (10±0.5)·10-3mol/l, after which the mixture is incubated before the establishment of full adsorption equilibrium and produce bionanocomposite method of magnetic separation, followed by centrifugation and washing, and to highlight heteronuclei sequence in a solution containing nanoparticles of cobalt ferrospinel associated with synthetic single-stranded-oligonucleotide 5'-dGndTmwhere n=5 to 30, m=10-35, and sodium phosphate at a concentration of (10±0.5)·10-3mol/l, add molecules of the oligonucleotide adapter containing the sequence of the oligo-dAxon the 3'-end and a site at the 5'end, complementary to the specific heteronuclei sequence in solution, after which the mixture is again incubated before the establishment of full adsorption equilibrium and produce bionanocomposite method of magnetic separation, followed by centrifugation and washing.

3. The method according to p. 2, namely, that n preferably equals = 10 to 25, and m is preferably 15-25.



 

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4 cl 7 dwg

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology, in particular to short interfering RNA (siRNA), and can be used in antitumor therapy. On the basis of genome analysis the sequences of siRNA against the human gene HIF1A with SEQ ID NO:1-2 are designed, siRNA against the human gene HSP8A with SEQ ID NO:3-4, siRNA against the human gene APEX1 with SEQ ID NO:5-6, and siRNA against the human gene CCND3 with SEQ ID NO:7-8, associated with cell proliferation of human pancreatic adenocarcinoma. Using the obtained siRNA, including as part of a lentiviral vector, results in suppression of cell proliferation of human pancreatic adenocarcinoma and tumor destruction. The invention enables to achieve the level of suppression of proliferation up to 65% using siRNA to genes HIF1A and HSP8A associated with stress and responsible for the major proliferative (CCND3) and reparative (APEX-1) ways of cell division.

EFFECT: improving level of suppression of proliferation.

3 cl, 10 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and microbiology and represents a method for identifying a cluster of antigens coding staphylococcal proteins that are exotoxins. The present method is implemented by performing a single multiple-primer polymerase chain reaction in two reaction mixtures, first of which contains primers to genes coding such staphylococcal proteins, as thermonuclease, beta-glucosidase in the species S.aureus, S.epidermidis, S.haemolyticus, S.lugdunensis, S.saprophyticus, and the second one - to set1, set2, set3, set4, set5 genes coding the exotoxins. That is followed by a comparative analysis of amplified gene fragments prepared in two sample aliquots and a positive control reference according to the identification table. The present invention also discloses a test system for implementing the above method, which contains DNA recovery components, PCR components, and result analysis components. The PCR components contain a 10-merous buffer solution, pH 8.4, deionised sterile water, one positive control reference, Taq polymerase, mixture of four dNTPs, mixture of primers No.1 containing epi-F, epi-R, aur-F, aur-R, hae-F, hae-R, lug-F, lug-R, sap-F, sap-R in a ratio of 1:1:1:1:1:1:1:1:1:1, and a mixture of primers No. 2 containing set1-F, set1-R, set2-F, set2-R, set3-F, set3-R, set4-F, set4-R, set5-F, set5-R in a ratio of 1:1:1:1:1:1:1:1:1:1.

EFFECT: invention enables recovering the cluster of genes coding the staphylococcal proteins, a molecular weight of which makes 25 to 35 kD consisting of almost 200 amino acid residues and having a tertiary structure high-homologous with some staphylococcus superantigens (enterotoxins, TSST-1 toxins) and with pyrogenous streptococcal exotoxin C.

2 cl, 3 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: invention is a recombinant plasmid DNA pET3.54, encoding the polypeptide FN3.54, interacting with human tumour necrosis factor. The present invention also discloses the recombinant strain of bacteria Escherichia coli BL21(DE3)/pET3.54 - producer of the polypeptide FN3.54, interacting with human tumour necrosis factor.

EFFECT: invention enables to obtain the desired protein with high yield, using a smaller amount of the inducer and the expression level of total cell protein.

2 cl, 4 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: method can be applied for processing of liquid wastes of obtaining galvanic and chemical coatings with cobalt. To obtain cobalt(II)-ammonium phosphates water solution, which contains cobalt(II), phosphate and ammonium is prepared, and as source of cobalt(II) applied is liquid wastes of obtaining coatings with cobalt -waste solution of galvanic cobalt plating and/or waste solution of chemical cobalt plating.

EFFECT: method makes it possible to obtain chemical products, applied in industry, agriculture with low prime cost.

11 cl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to chemistry of platinum metals, particularly synthesis of palladium compounds, specifically synthesis of heteronuclear acetates of palladium with non-ferrous metals. The method of producing heteronuclear acetates of palladium with non-ferrous metals involves reaction of an acetate compound of palladium and a non-ferrous metal compound in a glacial acetic acid solution, where the reaction of compounds, taken in molar ratio palladium: non-ferrous metal of 1:(0.90-0.97), takes place in glacial acetic acid used in amount of (600-800)% of the molar amount of palladium, at temperature (70-90)°C with evaporation of the solvent to wet or dry residue, with repeated addition of glacial acetic acid in amount of (200-600)% of the molar amount of palladium, repeated evaporation of the solvent at temperature (80-120)°C, with treatment of the dry residue, pre-heated to (70-90)°C, with a solution of a mixture of benzene or toluene and acetic acid anhydride with volume ratio thereof equal to (4-8):1 respectively, the amount of the acetic acid anhydride being equal to (20-60)% of the molar amount of palladium, at temperature (70-100)°C for (2-30) minutes, cooling the obtained suspension to temperature (40-70)°C and filtering the desired compound. In another version, the method involves reaction of a palladium acetate and an acetate compound of a non-ferrous metal in glacial acetic acid solution with solvent evaporation, where the reaction of compounds, taken in molar ratio palladium: non-ferrous metal equal to 1:(0.90-0.97), takes place in glacial acetic acid used in amount of (400-600)% of the molar amount of palladium, at temperature (80-120)°C with solvent evaporation to a dry residue, with subsequent treatment thereof with a solution of a mixture of benzene or toluene and acetic acid anhydride, pre-heated to (70-90)°C, with volume ratio thereof equal to (4-8):1 respectively, the acetic acid anhydride being in amount of (20-60)% of the molar amount of palladium, at temperature (70-100)°C for (2-30) minutes, cooling the obtained suspension to temperature (40-70)°C and filtering the desired compound.

EFFECT: invention enables to realise a simple and stable method of producing desired compounds with high output.

4 cl, 46 ex, 2 tbl

FIELD: process engineering.

SUBSTANCE: invention may be used in powder metallurgy. Proposed powder of oxide or mix of oxides of tungsten or cobalt is produced by neutralising water solution of appropriate inorganic salt or salts in the presence of black pre-added to solution in amount of MeO : C = 1 : (2-5) in terms of oxide or mix of oxides WO3 and/or Co3O4. Conversion into tungsten-carbide is performed by microwave radiation with frequency of 2450-3000 MHz at 700-1200 W.

EFFECT: production of tungsten and cobalt carbide powders with specific surface approximating to 6,93 m2/g and particle size of 100-400 nm.

2 cl, 3 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: magnetic cobalt-manganese sulphide with giant magnetoresistance contains manganese, sulphur and cobalt in the following ratio, wt %: cobalt 10-20, manganese 40-30, sulphur 50.

EFFECT: invention enables to design microelectronic elements based on the giant magnetoresistance effect for a wide range of temperature and magnetic fields, cuts expenses on manufacturing materials with giant magnetoresistance.

2 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention refers to the field of metallurgy, particularly to the method for preparation of cobalt aluminate used for surface modification of the molded pieces made of heat-resistant alloys. The mixture of aluminium oxide, cobalt oxide and aqueous cobalt carbonate powders taken at following component ratio (wt %): cobalt oxide 25-40, aqueous cobalt carbonate 3-25, aluminium oxide - the rest, is calcinated and pulverised.

EFFECT: invention allows to reduce the materials cost.

4 ex

FIELD: chemical industry; other industries; production of the magnetic materials and the catalysts on the basis of ferrites- chromites of cobalt (II).

SUBSTANCE: the invention is pertaining to the method of production of the solid solutions of CoFe2-xCrxO4 composition with the spinel structure and may be used in chemical industry for production of the magnetic materials and the catalysts on the basis of ferrites- chromites of cobalt (II). The method of production of the solid solutions of CoFe2-xCrxO4 composition includes homogenization of the source oxides of cobalt (II), iron (III), chromium(chrome) (III), briquetting and the thermal treatment at the temperature of 800-1000°С. The homogenization is conducted in the presence of the mineralizer, in the capacity of which use the mixture of 0.3-0.5 mass % of potassium chloride and 0.3-0.5 mass % of sodium chloride. The technical result of the invention is reduction of the time of the production process, the decreased power input and the production price.

EFFECT: the invention ensures reduction of the time of the production process, the decreased power input and the production price.

2 ex, 2 tbl

The invention relates to a technology of inorganic substances, in particular to methods for cobalt (II) sulfate of cobalt containing material

The invention relates to the field of chemical-technological purposes, such as salts of cobalt, used in industry for the production of paints and enamels, catalysts, batteries

FIELD: chemistry.

SUBSTANCE: method of obtaining ultradispersive powders of carbonates includes carbonisation of an initial raw material water suspension under conditions of an increase of carbon dioxide pressure with the simultaneous suspension homogenisation. As the initial raw material used are coarsely dispersive powders of respective carbonates of iron, or calcium, or magnesium, or calcium-magnesium, or calcium-iron-magnesium. The carbonisation process is carried out at a temperature of 6-20°C and a short-term to 1 s increase of pressure from 2.6 to 3.0 MPa. A solution of unstable hydrocarbonates is discharged, filtered and subjected to thermal processing at a temperature of 105°C.

EFFECT: invention makes it possible to simplify obtaining ultradispersive carbonates.

1 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: method of obtaining the solid magnesium material Sm2Fe17Nx includes mixing Sm and Fe powders, their mechanical activation and the following nitration. First, mechanical activation in a high-energy-voltage grinder is carried out in the inert atmosphere without content of moisture for 2-3 hours. For nitration in the grinder reactor introduced is ammonia and hydrogen in a ratio of NH3 - 85-95%, H2 - 5-15% and mechanical activation is continued for 5-7 hours. After that, a highly-molecular compound polymethylmetacrylate (PMMA) is introduced in a quantity of 2-4% of the weight of the initial powder mixture and the process of mechanical activation is continued for than 10-15 minutes.

EFFECT: invention makes it possible to reduce the time of obtaining the solid magnesium material and increase its coercive force.

1 tbl

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