RU2001120905A

RU2001120905A - Composition for immobilization of biological macromolecules in hydrogels, method for preparing the composition, biochip, method for PCR on biochip

Info

Publication number: RU2001120905A
Application number: RU2001120905/04A
Authority: RU
Inventors: Андрей Дарьевич Мирзабеков; Алла Юрьевна Рубина; Сергей Васильевич Паньков; Александр Николаевич Перов; Валентина Владимировна Чупеева
Original assignee: Институт молекулярной биологии им. В.А. Энгельгардта РАН
Filing date: 2001-07-25
Publication date: 2003-09-20

Claims

1. Composition (K) for immobilization of biological macromolecules in hydrogels by copolymerization of the following composition:

K = aA + bB + cC + dD + eE,

including A - monomer based on derivatives of acrylic and methacrylic acids;

B is a water soluble crosslinking agent;

C is a modified biological macromolecule containing

unsaturated group;

D - water-soluble compound as a component of the medium

copolymerization;

E is water

where a, b, c, d, e is the percentage (X) of each component in the composition (X = m / v × 100% for solids and X = v / v × 100% for liquid substances), in which the total content the monomer and the crosslinking agent lies in the range of 3-40% (3≤ (a + b) (40), the ratio of the monomer and the crosslinking agent is in the range 97: 3-60: 40, and the percentage of components C, D and E is in within the limits of 0.0001% ≤с≤10%; 0% ≤d≤90%; ≤5% ≤ е≤95%, used for manufacturing biochips.

2. The composition according to claim 1, containing as monomers (A) acrylamide, methacrylamide, N- [tris (hydroxymethyl) methyl] acrylamide, 2-hydroxyethyl methacrylate.

3. The composition according to claim 1, in which the monomers are used individually or in a mixture.

4. The composition according to claim 1, containing as a crosslinking agent (B) N, N'-methylenebisacrylamide, N, N'-ethylenebismethacrylamide, N, N '- (1,2-dihydroxyethylene) bisacrylamide, polyethylene glycol diacrylate.

5. The composition according to claim 1, in which the crosslinking agents are used individually or in a mixture.

6. The composition according to claim 1, in which an oligonucleotide of the general formula I is used as an immobilized modified biological macromolecule (C)

where OLIGO is an oligonucleotide;

R ¹ , R ² , R ³ - H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Z is (CH ₂ ) _n CH (CH ₂ OH) CH ₂ OX, where n = 1-6; or (CH ₂ ) _n —OX, where n = 2-6;

X is a phosphodiester group linking the unsaturated fragment to the 5 ′ and / or 3 ′ end of the oligonucleotide.

R ⁴ is H, (CH ₂ ) _n OH, where n = 2-6;

Y- (pC ₆ H ₄ ) _n , where n = 0-2.

7. The composition according to claim 1, in which the DNA of general formula II is used as an immobilized modified biological macromolecule (C)

5’-X-O-DNA-3’-O-Z,

where DNA is a DNA fragment,

X = H or H ₂ PO ₃ , Z = -CO-Y-CR ¹ = CR ² R ³

or

X = —CO — Y — CR ¹ CR ² R ³ , Z H or H ₂ PO ₃ ,

R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2.

8. The composition according to claim 1, in which the DNA of general formula III is used as an immobilized modified macromolecule (C)

where DNA = DNA fragment;

R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₃ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2.

9. The composition according to claim 1, in which the DNA of general formula IV is used as an immobilized modified macromolecule (C)

where DNA = DNA fragment;

R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2;

R ⁴ = H, (CH ₂ ) _n OH, where n = 2-6;

Z = - (CH ₂ ) _n CH (CH ₂ OH) CH ₂ OX, where n = 1-6, or - (CH ₂ ) _n OX, where n = 2-6;

X = phosphodiester group linking the unsaturated fragment to the 5’ and / or 3’ end of the DNA fragment.

10. The composition according to claim 1, in which a protein of general formula V is used as an immobilized modified biological macromolecule (C)

where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

X = NH, O, CH ₂ , S;

Y = (pC ₆ H ₄ ) _n , where n = 0-2;

R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20.

11. The composition according to claim 1, in which a protein of general formula VI is used as an immobilized modified biological macromolecule (C)

where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , n = 0-2;

X = NH, O, S, CH ₂

R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20;

W = NH, O, CH ₂ ;

F = (CH ₂ ) _n , n = 1, 2;

Z = NH, S.

12. The composition according to claim 1, in which a protein of general formula VII is used as an immobilized modified biological macromolecule (C)

where R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20.

13. The composition according to claim 1, where a water-soluble high-boiling organic compound is used as a component of the copolymerization (D) medium.

14. The composition of claim 13, wherein N, N-dimethylformamide and dimethyl sulfoxide are used as the water-soluble high boiling organic compound.

15. The composition according to claim 1, characterized in that as a component of the medium for photoinduced polymerization using a water-soluble polyhydroxyl compound.

16. The composition according to p. 15, characterized in that glycerol, sucrose and polyvinyl alcohol are used as a water-soluble polyhydroxyl compound.

17. The method of preparing the composition (K) according to claim 1 for the immobilization of biological macromolecules in hydrogels by copolymerization in a mixture of the following composition:

K = aA + bB + cC + dD + eE

including

A is a monomer based on derivatives of acrylic and methacrylic acids;

B is a water soluble crosslinking agent;

C is a modified biological macromolecule containing

unsaturated group;

D - water-soluble compound as a component of the medium

copolymerization;

E is water

where a, b, c, d, e is the percentage (X) of each component in the composition (X = m / v × 100% for solids and X = v / v × 100% for liquid substances), in which the total content the monomer and the crosslinking agent is in the range of 3-40% (3 <(a + b) <40), the ratio of the monomer and the crosslinking agent is in the range of 97: 3-60: 40, and the percentage of components C, D and E is in within the limits of 0.0001% ≤с≤10%; 0% ≤d≤90%; 5% ≤ е≤95%, used for manufacturing biochips.

18. Modified DNA fragments of the following structure:

5’-X-O-DNA-3’-O-Z,

where DNA is a DNA fragment,

X — H or H ₂ PO ₃ , Z = —CO — Y — CR ¹ = CR ² R ³ , where R ¹ , R ² , R ³ —H, alkyl C ₁ —C ₆ , Ph, PhCH ₂ -;

or X = —CO — Y — CR ¹ = CR ² R ³ , Z = H or H ₂ PO ₃ , where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2;

obtained by direct acylation of DNA fragments with unsaturated acid anhydrides,

or

where DNA = DNA fragment;

R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2.

obtained by reductive amination of apurinated DNA followed by acylation of the amino derivative with activated esters of unsaturated acids, or

where DNA = DNA fragment;

R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2;

R ⁴ = H, (CH ₂ ) _n OH, where n = 2-6;

X = phosphodiester group linking the unsaturated fragment to the 5’ and / or 3’ end of the DNA fragment,

obtained by PCR amplification using a synthetic primer carrying an unsaturated group at the 5 ′ or 3 ′ end.

19. Modified proteins of the following structure:

where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

X = NH, O, CH ₂ , S;

Y = (pC ₆ H ₄ ) _n , where n = 0-2;

R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20,

obtained by acylation of free amino groups of a protein with activated esters of unsaturated acids

or

where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (p-C ₆ H ₄ ) _n , n = 0-2;

X = NH, O, S, CH ₂ .

R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20;

W = NH, O, CH ₂ ;

F = (CH ₂ ) _n , n = 1, 2;

Z = NH, S;

obtained by alkylation of a sulfhydryl or amino group of a protein with derivatives of α, β-unsaturated and α-halocarbonyl compounds,

or

where R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20,

by treating a recombinant protein containing a terminal His-6 region with methacrylamide derivatives of nitrilotriacetic acid in the presence of Ni (II) salts.

20. Modified porous glass (CPG) of the following structure:

where R ¹ , R ² = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

R ³ = C ₁ -C ₆ alkyl;

Y = (pC ₆ H ₄ ) _n , where n = 0-2,

as a carrier for introducing the unsaturated acid residue at the 3'-end of the oligonucleotides of formula I according to claim 6 under conditions of automatic solid-phase synthesis.

21. Activated esters of the following structure:

where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (pC ₆ H ₄ ) _n , where n = 0-2;

Z = NH, O, CH ₂ , S

R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , where n = 1-20;

X = succinimidiooxy, n - nitrophenoxy, pentafluorophenoxy or any other acceptor good leaving group as a modifying agent to produce a protein of formula V.

22. Carbonyl compounds of the following structure:

where R ¹ , R ² , R ³ = H, alkyl C ₁ -C ₆ , Ph, PhCH ₂ -;

Y = (p-C ₆ H ₄ ) _n , n = 0-2;

X = NH, O, S, CH ₂ ;

R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20;

W = NH, O, CH ₂ ,

Z = halogenomethyl, vinyl, or any other fragment containing an active multiple bond, as a modifying agent to produce a protein of formula VI.

23. Methacrylamide derivatives of nitrilotriacetic acid of the general formula:

CH ₂ = C (Me) —CO — NH — R — CH (COOH) —N (CH ₂ COOH) ₂ ,

where R = (CH ₂ ) _n , (CH ₂ CH ₂ O) _n , n = 1-20,

as a modifying agent to obtain a protein of the formula VII.

24. The biochip made on the basis of the composition according to claim 1, in which the gel layer formed on the substrate is separated by empty spaces into several cells, each cell may or may not contain immobilized macromolecules, and macromolecules immobilized in different cells may vary in nature and properties.

25. The biochip according to paragraph 24, in which these cells form a regular one-dimensional or two-dimensional structure (array).

26. The biochip according to paragraph 24, in the manufacture of which the polymerization mixture is applied to the substrate using an automatic device (robot) equipped with one or more microdisperses.

27. The biochip according to claim 26, the manufacture of which uses rod-type microdispersers.

28. The biochip according to claim 26, in the manufacture of which non-contact jet microdisperses are used.

29. The biochip according to claim 26, in the manufacture of which several microdispersers are used that form a regular structure.

30. The biochip according to paragraph 24, in the manufacture of which one or more substrates with droplets of the polymerization mixture deposited on them during polymerization are placed in an airtight container in an oxygen-free inert atmosphere with controlled humidity.

31. The biochip according to paragraph 24, in the manufacture of which the container is filled with one of the following gases: N ₂ , Ar, CO ₂ .

32. The biochip according to paragraph 24, in the manufacture of which the gas medium in the container with the substrates is continuously or periodically updated.

33. The method of performing PCR on a biochip according to paragraph 24 by adding an amplification solution, direct (F) and reverse (R) primers and test nucleic acid samples and incubating the biochip under conditions of cyclic temperature changes that allow for PCR amplification.

34. The method of PCR on the biochip according to paragraph 24 by incubating the biochip at a constant temperature with a hybridization solution containing the studied nucleic acid samples, for their hybridization with immobilized primers (synthetic oligonucleotides), incubation of the biochip containing nucleic acids hybridized with immobilized primers, amplification solution containing direct (F) and reverse (R) primers, at a constant temperature, replacing the amplification solution outside the gel elements of the biochip hydrophobic Second fluid (mineral oil), which completely isolates the biochip cell from each other, and incubating the biochip under conditions of cyclic temperature changes that give effect to PCR amplification.