Attachment of oligonucleotides to solid supports through schiff base type linkages for capture and detection of nucleic acids

• US 20020081591A1
• Filed: 02/09/2001
• Published: 06/27/2002
• Est. Priority Date: 07/29/1999
• Status: Active Grant

• Current Assignee: Eugeny Alexander Lukhtanov, Joel Hedgpeth, Mikhail A. Podyminogin
• Original Assignee: Eugeny Alexander Lukhtanov, Joel Hedgpeth, Mikhail A. Podyminogin
• Inventors: Podyminogin, Mikhail A., Lukhtanov, Eugeny Alexander, Hedgpeth, Joel
• Granted Patent: US 6,548,652 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support

Abstract

Derivatized oligonucleotides (ODNs) are coupled to a solid support in improved yield resulting in a high density of coupled oligonucleotide per surface unit of the support, through a Schiff base type bond formed between an NH₂ group attached either to the solid support or to the ODN and an aromatic aldehyde attached to the other of the solid support and the ODN. The preferred solid support-ODN conjugate is formed between semicarbazide groups attached to a glass surface and an aromatic aldehyde attached at either 3′, or 5′ end of an ODN or to an intermediate nucleotide of the ODN.

Claims

1. A composition of matter comprising an oligonucleotide attached to a solid support, having the formula selected from the group consisting of formula (i) and formula (ii),

2. A composition of matter in accordance with claim 1 in accordance with formula (i).

3. A composition of matter in accordance with claim 2 where X is —

NH—

C═

(O)—

NH—

N═

or —

C═

(O)—

NH—

N═

.

4. A composition of matter in accordance with claim 3 where X is —

NH—

C═

(O)—

NH—

N═

.

5. A composition of matter in accordance with claim 2 where Q represents a benzene ring.

6. A composition of matter in accordance with claim 4 where Q represents a benzene ring.

7. A composition of matter in accordance with claim 2 where the solid phase is a glass surface.

8. A composition of matter in accordance with claim 1 in accordance with formula (ii).

9. A composition of matter in accordance with claim 8 where X is —

NH—

C═

(O)—

NH—

N═

or —

C═

(O)—

NH—

N═

.

10. A composition of matter in accordance with claim 9 where X is —

NH—

C═

(O)—

NH—

N═

.

11. A composition of matter in accordance with claim 8 where Q represents a benzene ring.

12. A composition of matter in accordance with claim 8 where the solid phase is a glass surface.

13. A composition of matter in accordance with claim 2 where NH₂ groups not attached to the oligonucleotide are end-capped with a covalently linked moiety containing an anion.

15. A composition of matter in accordance with claim 14 where n is 3.

16. A composition of matter in accordance with claim 14 where R₃ is H.

17. A composition of matter in accordance with claim 14 where the phenyl group is attached through linker V to the 5′

-end of the oligonucleotide.

18. A composition of matter in accordance with claim 14 where the phenyl group is attached through linker V to the 3′

-end of the oligonucleotide.

19. A composition of matter in accordance with claim 14 where n is 3, R₃ is H, and the phenyl group is attached through linker V to the 5′

-end of the oligonucleotide.

20. A composition of matter in accordance with claim 19 where the linker V includes an OP(O)(O⁻

)O—

moiety.

14. A composition of matter comprising an oligonucleotide attached to a glass support, having the formula

21. A method of attaching an oligonucleotide to a solid support, to provide a solid-support oligonucleotide conjugate, the method comprising the step of:

reacting a derivatized solid support of the formula (iii) with a derivatized oligonucleotide of the formula (iv), or by reacting a derivatized solid support of the formula (v) with a derivatized oligonucleotide of the formula (vi)

 

where the symbol solid-phase represents a solid matrix;

n=1 to 30;

m is 1 to 30;

R₃ is H, C₁-C₆alkyl or C₃-C₆cycloalkyl;

X is —

N═

, —

UN═

, —

C═

(U)—

NH—

N═

, —

NH—

C═

(U)—

NH—

N═

, —

NH—

NH—

CH═

(U)—

NH—

N═

or —

U—

NH—

C═

(U)—

NH—

N═

, where U is independently O or S;

Q is carbocyclic condensed or not-condensed aromatic ring, or a condensed or not-condensed heteroaromatic ring said carbocyclic or heteroaromatic ring being optionally substituted with a lower alkyl, lower alkoxy or halogen group;

V is a linker having the length of 2 to 100 atoms, that contains carbon to carbon bonds and optionally and independently may contain carbon to oxygen bonds and one or more moieties selected from the group consisting of —

NH—

, —

OH, —

O—

, —

NH—

C(═

O)—

, —

C═

(O)—

NH—

, —

NH—

C(═

O)—

NH—

, —

NH—

C(═

S)—

NH—

, —

S—

, OP(O)(O⁻

)O— and

—

S—

S—

;

W is a linker having the length of 2 to 100 atoms, that contains carbon to carbon bonds and optionally and independently may contain carbon to oxygen bonds and one or more moieties selected from the group consisting of —

NH—

, —

OH, —

O—

, —

NH—

C(═

O)—

, —

C═

(O)—

NH—

, —

NH—

C(═

O)—

NH—

, —

NH—

C(═

S)—

NH—

, —

S—

, OP(O)(O⁻

)O— and

—

S—

S—

, said linker W terminating with a carbon atom adjacent to X;

T is a valence bond or a linker having the length of 1 to 100 atoms, that contains carbon to carbon bonds and optionally and independently may CONTAIN carbon to oxygen bonds and one or more moieties selected from the group consisting of —

NH—

, —

OH, —

O—

, —

NH—

C(═

O)—

, —

C═

(O)—

NH—

, —

NH—

C(═

O)—

NH—

, —

NH—

C(═

S)—

NH—

, —

S— and

—

S—

S—

, said linker T terminating with a carbon atom adjacent to X.

R₅ is —

O—

P═

(O)(—

U⁻

)-3′

-oligomer of nucleotides or —

O—

P═

(O)(—

U⁻

)-5′

-oligomer of nucleotides where U is O or S.

22. A method in accordance with claim 21 where a derivatized solid support of the formula (iii) is reacted with a derivatized oligonucleotide of the formula (iv).

23. A method in accordance with claim 22 where the reaction is conducted in an aqueous phase having a pH less than approximately 8.

24. A method in accordance with claim 23 where the reaction is conducted in an aqueous phase having a pH less than approximately 7.

25. A method in accordance with claim 22 where X is selected from the group consisting of —

NH—

C═

(O)—

NH—

N and —

N.

26. A method in accordance with claim 22 R₃ is H and Q represents a benzene ring.

27. A method in accordance with claim 21 where a derivatized solid support of the formula (v) is reacted with a derivatized oligonucleotide of the formula (vi).

29. A derivatized solid support in accordance with claim 28 that is in accordance with formula (iii).

30. A derivatized solid support in accordance with claim 29 where X is selected from the group consisting of —

C═

(O)—

NH—

N═ and

—

NH—

C═

(O)—

NH—

N═

.

28. A derivatized solid support of formula (iii) or of formula (v)

31. A phosphoramidite reagent of the formula

32. A phosphoramidite reagent in accordance with claim 31 where R₃ is H.

33. A phosphoramidite reagent in accordance with claim 32 where Q represents a benzene ring.

35. A derivatized controlled pore glass support in accordance with claim 34 where R₃ is H.

36. A derivatized controlled pore glass support in accordance with claim 35 where n is 4 and R₂ is CH₃CO—

.

39. The method of claim 38 where the Schiff base is formed between a semicarbazide group attached to the solid surface and an aromatic aldehyde attached to each of the oligonucleotides.

40. The method of claim 39 where the solid surface is a glass surface.

34. A derivatized controlled pore glass support of the formula

37. A method of coupling a solid support to an oligonucleotide, the support having a matrix and NH—

C(O)—

NHNH₂ groups covalently attached to the matrix through a covalently attached linker group, the oligonucleotide including cytosine nucleosides, the method comprising the step of;

reacting in an aqueous phase in the presence of bisulfite the solid support with the oligonucleotide.

38. A method of binding a nucleic acid or a fragment thereof to a substantially complementary strand of oligonucleotide, said method comprising the steps of:

providing an array of oligonucleotides of varying sequences on a solid support surface where each ODN is bound to the surface by a covalent bond including a Schiff base formed between an NH₂ group and an aromatic aldehyde, and

contacting the nucleic acid or fragment thereof with the array of oligonucleotides bound to the solid surface.