Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports

• US 20030120035A1
• Filed: 03/13/2002
• Published: 06/26/2003
• Est. Priority Date: 03/14/2001
• Status: Active Grant

• Current Assignee: University of Houston (University of Houston System)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Gao, Xiaolian, Zhang, Hua, Zhou, Xiaochuan, Leproust, Eric, Xiang, Qin, Yu, Peilin, Pellois, Jean Philippe
• Granted Patent: US 7,211,654 B2
• Time in Patent Office: Days
• US Class Current: 530/333
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

A method of modulation of synthesis capacity on and cleavage properties of synthetic oligomers from solid support is described. The method utilizes linker molecules attached to a solid surface and co-coupling agents that have similar reactivities to the coupling compounds with the surface functional groups. The preferred linker molecules provide an increased density of polymers and more resistance to cleavage from the support surface. The method is particularly useful for synthesis of oligonucleotides, oligonucleotides microarrays, peptides, and peptide microarrays. The stable linkers are also coupled to anchor molecules for synthesis of DNA oligonucleotides using on support purification, eliminating time-consuming chromatography and metal cation presence. Oligonucleotides thus obtained can be directly used for mass analysis, DNA amplification and ligation, hybridization, and many other applications.

Claims

1. A composition comprising a selectively cleavable linker for polymer synthesis comprising a linker group having first and second ends, wherein said first end comprises a substrate attaching group and wherein said second end comprises a polymer attaching group, wherein said polymer attaching group is covalently linked to an anchor moiety.

2. The composition of claim 1, wherein said linker group is selected from the group consisting of an alkyl group, a polyethyleneglycol and an amide group.

3. The composition of claim 2, wherein said alkyl group is —

(CH₂)_n—

, wherein n is from about 4 to about 20.

4. The composition of claim 2, wherein said polythylene group is —

(OCH₂CH₂)_n—

, wherein n is from about 1 to about 20.

5. The composition of claim 1, wherein said substrate attaching group is selected from the group consisting of chlorosilyl and alkyloxysilyl functional groups.

6. The composition of claim 1, wherein said polymer attaching group is selected from the group consisting of amine, hydroxyl, thiol, carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanate.

7. The composition of claim 1, wherein said linker is covalently bound to a support.

8. The composition of claim 7, wherein said support is selected from the group consisting of polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, nitrocellulose and nylon membranes, polytetraflouroethylene, polyvinylidendiflouride, polystyrene, and polycarbonate.

9. The composition of claim 1, wherein said anchor moiety is selected from anchor moieties having the following structure:

10. The composition of claim 9, wherein said anchor moiety is selectively cleavable.

11. The stable linker of claim 10, wherein said anchor moiety is cleavable by 2-OH assisted 1-phosphate hydrolysis.

12. The composition of claim 9, wherein said anchor moiety includes a synthesis initiation site.

13. The composition of claim 12, further comprising a polymer attached to said synthesis initiation site.

14. The composition of claim 13, wherein said polymer is selected from group consisting of a polypeptide and a oligonucleotide.

15. A compound of the structure:

Rs-L-Rp

wherein R_s is a substrate attaching group, R_p is a polymer attaching group, and L is the linker.

16. The compound of claim 15, wherein said substrate attaching group is selected from the group consisting of chlorosilyl and alkyloxysilyl functional groups.

17. The compound of claim 15, wherein said polymer attaching group is selected from the group consisting of amine, hydroxyl, thiol, carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanate.

18. A compound having the following structure:

S—

R₁—

R₂—

R₃—

R₄

wherein S is a substrate, R₁ is a linker group, R₂ is a polymer, R₃ is an anchor, and R₄ is either nothing or a polymer, where R₂ and R₄ do not need to be the same.

19. The compound of claim 18, wherein said linker is selected from group consisting of akyl linkers, polyethyleneglycol, amide, and alkylenyl linkers.

20. A compound of the structure:

Rs-L-Rp

wherein R_s is a substrate attaching group, R_p is a polymer attaching group, and L is the linker.

21. The compound of claim 20, wherein said substrate attaching group is selected from the group consisting of chlorosilyl and alkyloxysilyl functional groups.

22. The compound of claim 20, wherein said polymer attaching group is selected from the group consisting of amine, hydroxyl, thiol, carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanate.

23. The compound of claim 22, wherein B is uridine.

24. The compound of claim 20, wherein said polymer is an oligonucleotide.

25. A method for synthesizing oligonucleotides comprising:

a) providing

i) a substrate;

ii) a plurality of stable linkers;

iii) a plurality of anchor moieties; and

iv) nucleotide monomers;

b) derivitizing said substrate with said plurality of stable linkers;

c) attaching said anchor moieties to said stable linkers; and

d) synthesizing a oligonucleotide on said plurality of anchor moieties from said monomers.

26. A method for synthesizing oligonucleotides comprising:

a) providing

i) a substrate;

ii) a plurality of stable linkers;

iii) a plurality of anchor moieties; and

iv) nucleotide monomers;

b) derivitizing said substrate with said plurality of stable linkers;

c) synthesizing a oligonucleotide on said plurality of anchor moieties from said monomers; and

d) attaching said anchor moieties to said polymers; and

e) synthesizing a oligonucleotide on said plurality of anchor moieties from said monomers.

27. A method for controlling the number of oligonucleotides synthesized at a predetermined site on a substrate comprising:

a) Providing

i) a substrate;

ii) a plurality of stable linkers;

iii) a plurality of anchor moieties;

iv) nucleotide monomers; and

v) co-coupling agents;

b) derivitizing said substrate with said plurality of stable linkers;

c) attaching said anchor moieties to said stable linkers; and

d) synthesizing a oligonucleotide on said plurality of anchor moieties from said monomers and co-coupling agents

28. A method of providing purified oligonucleotides comprising:

a) providing;

i) a substrate comprising oligonucleotides attached to a substrate via an anchor moiety attached to a stable linker group;

ii) a deprotecting solution; and

iii) and a wash solution;

b) deprotecting said oligonucleotides with said deprotecting solution,

c) washing said oligonucleotides attached to a substrate with said wash solution,

d) cleaving said oligonucleotides at said anchor group to provide purified oligonucleotides, wherein said purified oligonucleotides are characterized by the substantial absence of small molecules formed during deprotection step.

29. A method of providing purified oligonucleotides comprising:

a) providing;

i) a substrate comprising oligonucleotides attached to a substrate via an anchor moiety attached to an oligonucleotide attached a stable linker group;

ii) a deprotecting solution; and

iii) a wash solution;

b) deprotecting said oligonucleotides with said deprotecting solution,

c) washing said oligonucleotides attached to a substrate with said wash solution,

d) cleaving said oligonucleotides at said anchor group to provide purified oligonucleotides, wherein said purified oligonucleotides are characterized by the substantial absence of small molecules formed during deprotection step.

30. A method of providing purified oligonucleotides for use of enzymatic reactions:

a) providing;

i) oligonucleotides obtained from solid phase synthesis and directly purified while the oligonucleotides attached on the support; and

ii) oligonucleotides having 5′

-OH or 5′

-phosphate, and 3′

-OH at the end of the sequence;

b) mixing oligonucleotides with a DNA polymerase and DNA template strands having a complementary sequence to the oligonucleotide at the 3′

-end in a DNA polymerization buffer; and

c) recovering copies of complementary DNA strands in the duplex with the template strand.

31. A method of providing purified oligonucleotides for use of enzymatic reactions:

a) providing;

i) oligonucleotides obtained from solid phase synthesis and directly purified while the oligonucleotides attached on the support; and

ii) oligonucleotides having 5′

-OH or 5′

-phosphate, and 3′

-OH at the end of the sequence;

b) mixing oligonucleotides with a DNA polymerase and DNA template strands having a complementary sequence to the oligonucleotide at the 3′

-end in a PCR buffer; and

c) recovering amplified copies of complementary DNA strands in the duplex with the template strand.

32. A method of providing purified oligonucleotides for use of enzymatic reactions:

a) providing;

i) oligonucleotides obtained from solid phase synthesis and directly purified while the oligonucleotides attached on the support; and

ii) oligonucleotides having 5′

-phosphate and 3′

-OH at the end of the sequence;

iii) oligonucleotides which in part form duplexes with dangling ends on both 3′

- and 5′

-end;

d) mixing oligonucleotides with a DNA ligase in a ligation buffer; and

e) recovering ligated DNA strands.

33. A method of providing purified oligonucleotides for use of enzymatic reactions:

a) providing;

i) oligonucleotides obtained from solid phase synthesis and directly purified while the oligonucleotides attached on the support; and

ii) oligonucleotides having 5′

-OH and 3′

-OH at the end of the sequence;

iii) oligonucleotides which in part form duplexes with dangling ends on both 3′

- and 5′

-end;

b) mixing oligonucleotides with a 5′

-kinase in a kinase buffer

c) recovering oligonucleotides having 5′

-phosphate and 3′

-OH at the end of the sequence

d) mixing oligonucleotides with a DNA ligase in a ligation buffer; and

e) recovering ligated DNA strands.

34. A compound having the following structure:

35. The compound of claim 34, wherein B is uridine.