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

• US 7,553,958 B2
• Filed: 03/21/2007
• Issued: 06/30/2009
• Est. Priority Date: 03/14/2001
• Status: Expired due to Term

• 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
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/726,269
• Publication Number:
  US 20070287832A1
• Time in Patent Office: 832 Days
• Field of Search: 435/6, 536/23.1, 536/25.3, 536/25.4
• US Class Current: 536/25.3
• 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 method of generating purified oligonucleotides comprising:

a) providing;

i) a substrate comprising an array of oligonucleotides attached to said substrate via anchor moieties attached to non-cleavable linkers attached to said substrate, wherein said anchor moieties comprise the structure —

C(X)—

C(Y), wherein X comprises —

OPO₂O—

, Y is a nucleophile and wherein said structure is part of a ring moiety;

ii) a deprotecting solution; and

iii) and a wash solution;

b) deprotecting said oligonucleotides with said deprotecting solution;

c) washing said oligonucleotides with said wash solution; and

d) cleaving said oligonucleotides at said anchor moieties to provide purified oligonucleotides.

2. The method of claim 1, wherein said deprotecting solution comprises ethyl diazoacetate (EDA) in ethanol.

3. The method of claim 1, wherein said anchor moieties are attached to said noncleavable linkers via a 5′

-O—

P—

linkage.

4. The method of claim 1, wherein said anchor moieties are attached to said oligonucleotides via a 3′

-O—

P—

O-3′

phosphodiester attachment.

5. The method of claim 1, wherein said cleaving comprises cleavage selected from the group consisting of chemical cleavage and enzymatic cleavage.

6. The method of claim 5, wherein said chemical cleavage comprises exposure to ammonium hydroxide.

7. The method of claim 5, wherein said enzymatic cleavage comprises exposure to a ribonuclease.

8. The method of claim 7, wherein said ribonuclease is selected from the group consisting of RNase A, T₁, and U₂.

9. The method of claim 7, wherein said ribonuclease cleaves said anchor 3′

-O—

P—

O-3′

nucleotide phosphodiester attachments.

10. The method of claim 1, wherein said oligonucleotides are further purified utilizing liquid chromotography.

11. The method of claim 1, wherein said oligonucleotides are further purified utilizing gel purification.

12. The method of claim 1, wherein said oligonucleotides comprise deoxyribonucleic acid.

13. The method of claim 1, wherein said oligonucleotides comprise ribonucleic acid.

14. The method of claim 1, wherein said oligonucleotides comprise a label.

15. The method of claim 14, wherein said label comprises a fluorescent label.

16. The method of claim 1, wherein said anchor moieties comprise ribose nucleotides.

17. Purified oligonucleotides generated according to the method of claim 1.

18. The oligonucleotides of claim 17, wherein said oligonucleotides comprise deoxyribonucleic acid.

19. The oligonucleotides of claim 17, wherein said oligonucleotides comprise ribonucleic acid.