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

• US 7,211,654 B2
• Filed: 03/13/2002
• Issued: 05/01/2007
• 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:
  US10/099,382
• Publication Number:
  US 20030120035A1
• Time in Patent Office: 1,875 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/25.3, 435/6
• US Class Current: 536/23.1
• 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 compound having the following structure:

S—

R₁—

R₂—

R₃—

R₄

wherein S is a substrate, R₁ is a non-cleavable linker group, R₂ an oligonucleotide, R₃ is an anchor, wherein said anchor comprises the structure —

C(X)—

C(Y), wherein X comprises —

OPO₂O—

, Y is a nucleophile and wherein said structure is part of a ring moiety, and R₄ is absent or is a polymer, where R₂ and R₄ are the same or different.

2. The compound of claim 1, wherein said linker is selected from group consisting of akyl linkers, polyethyleneglycol, amide, and alkylenyl linkers.

5. The compound of claim 1, wherein said polymer is a polynucleotide.

6. The compound of claim 5, wherein said polynucleotide comprises a label.

7. The compound of claim 1, wherein said polymer is a polyamino acid.

3. A method for synthesizing oligonucleotides comprising:

a) providing

i) a substrate;

ii) a plurality of non-cleavable linkers;

iii) a plurality of anchor moieties, 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; and

iv) nucleotide monomers;

b) derivatizing said substrate with said plurality of non-cleavable linkers;

c) attaching said anchor moieties to said non-cleavable linkers; and

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

4. A method for synthesizing oligonucleotides comprising:

a) providing

i) a substrate;

ii) a plurality of non-cleavable linkers;

iii) a plurality of anchor moieties, 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; and

iv) nucleotide monomers;

b) derivatizing said substrate with said plurality of non-cleavable linkers;

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

d) attaching said anchor moieties to said substrates; and

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

8. 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) derivatizing said substrate with said plurality of stable linkers;

c) attaching said anchor moieties to said stable linkers, wherein said anchor moieties are attached to said linkers via coupling a 5′

phosphoramidite present within said anchor moieties to said linkers through a 5′

-O—

P-linkage;

d) attaching said nucleotide monomers to said stable linkers attached to said anchor moeities through an anchor 3′

-O—

P—

O-3′

nucleotide attachment thereby creating a plurality of anchor 3′

-O—

P—

O-3′

nucleotide moeities; and

e) synthesizing oligonucleotides on said plurality of anchor-nucleotide moieties from said monomers,

wherein anchor 3′

-O—

P—

O-3′

nucleotide phosphodiester attachments are cleaved by a ribonuclease.

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

10. The method of claim 8, wherein cleaving with said ribonuclease produces, an oligonucleotide comprising a 3′

-OH.

11. The method of claim 8, wherein the 3′

and 5′

ends of the cleaved phosphodiester attachments are further modified using a 5′

or 3′

exonuclease.

12. The method of claim 11, wherein said 5′

or 3′

exonuclease removes a terminal phosphate group.