Methods for attachment of a polynucleotide to a preselected material

• US 6,022,714 A
• Filed: 12/19/1995
• Issued: 02/08/2000
• Est. Priority Date: 05/02/1985
• Status: Expired due to Term

• Current Assignee: Genetics Institute LLC (Pfizer Inc.)
• Original Assignee: Genetics Institute LLC (Pfizer Inc.)
• Inventors: Dougherty, Joseph P., Collins, Mary, Brown, Eugene L.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/574,860
• Time in Patent Office: 1,512 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.3, 435/91.5, 435/91.51, 435/91.52, 435/810, 436/501, 536/22.1, 536/23.1, 536/24.1, 536/24.3-24.33, 536/25.3, 935/77, 935/78, 935/16, 935/17, 935/88
• US Class Current: 435/91.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Compositions and methods for selectively linking a polynucleotide through its 5'"'"' or 3'"'"' end to one or more preselected materials such as insoluble matrices, solid supports, proteins, small molecular or labels are disclosed. Use of these compositions and methods in the production of diagnostic and affinity reagents are also disclosed.

Claims

1. A method for attaching a polynucleotide to a preselected material selected from the group consisting of a solid support, an insoluble matrix, a protein, a small molecule, and a label which comprises:

(a) attaching a first polynucleotide, through its 5'"'"' or 3'"'"' end, to a linking agent represented by the formula

space="preserve" listing-type="equation">(R.sub.1).sub.n R.sub.2 R.sub.3

wherein R₁ is a functional moiety selected from the group consisting of --COOH, --CHO, a detectable moiety, an affinity agent, and a hapten, wherein n is an integer from 1 to 20, wherein for n>

1, each of said R₁ may be a different selection from said group, R₂ is an aliphatic moiety, and R₃ is a phosphinylating or phosphorylating moiety represented by the formula ##STR13## wherein R₂ is selected from the group consisting of methyl, cyanoethyl and chlorophenyl;

R₅ is selected from the group consisting of morpholine, N,N-diisopropylamine and --OCH₂ CH₂ CN, and X is an oxygen atom, a sulfur atom or a pair of electrons;

wherein R₁ and R₂ are stable to conditions of polynucleotide synthesis, wherein said first polynucleotide is attached to said liking agent via R₃ moiety, and wherein said first polynucleotide and said linking agent together form an adapter molecule;

(b) ligating said adapter molecule with a second polynucleotide, through the 5'"'"' or 3'"'"' end of said second polynucleotide to the 5'"'"' or 3'"'"' end of the first polynucleotide not attached to the adapter molecule, to form a functionalized polynucleotide; and

(c) reacting said functionalized polynucleotide with said preselected material.

5. The method of claim 1 or 2 wherein R₁ is a functional moiety selected from the group further consisting of biotinyl and combinations thereof.

2. A method for attaching a polynucleotide to a preselected material selected from the group consisting of a solid support, an insoluble matrix, a protein, a small molecule, and a label which comprises:

(a) attaching a first polynucleotide, through its 5'"'"' or 3'"'"' end, to a linking agent represented by the formula

space="preserve" listing-type="equation">(R.sub.1).sub.n R.sub.2 R.sub.3

wherein R₁ is a functional group selected from the group consisting of --COOH, --CHO, a detectable moiety, an affinity agent, and a hapten, wherein n is an integer from 1 to 20, wherein for n>

1, each of said R₁ may be a different selection from said group, R₂ is an aliphatic moiety, and R₃ is a phosphinylating or phosphorylating moiety represented by the formula ##STR14## wherein R₄ is selected from the group consisting of methyl, cyanoethyl and chlorophenyl, R₅ is selected from the group consisting of morpholine, N,N-diisopropylamine and --OCH₂ CH₂ CN, and X is an oxygen atom, a sulfur atom or a pair of electrons;

wherein R₁ and R₂ are stable to conditions of polynucleotide synthesis, wherein said first polynucleotide is attached to said linking agent via the R₃ group, and wherein said first polynucleotide and said linking agent together form an adapter molecule;

(b) reacting said adapter molecule with said preselected material; and

(c) ligating said adapter molecule with a second polynucleotide, through the 5'"'"' or 3'"'"' end of said second polynucleotide to the 5'"'"' or 3'"'"' end of the first polynucleotide not attached to the adapter molecule.

3. A method for attaching a polynucleotide to a preselected material selected from the group consisting of a solid support, an insoluble matrix, a protein, a small molecule, and a label which comprises:

(a) attaching a first polynucleotide, through its 5'"'"' or 3'"'"' end, to a linking agent having a phosphoramidite or phosphate group, wherein said liking agent is selected from the group consisting of

12-methylcarboxyldodeclmethyl-N,N-diisopropylphosphoramidite;

5-oxohexylmethyl-N,N-diisopropylphosphoramidite;

12-phenyl-12-oxododecylmethyl-N-morpholinophosphoramidite;

12-methyl-11,12-diacetyltridecanediol methyl N-morpholinosphoramadite;

12-methylcarboxyldodecyl 2-chlorophenyl-2-cyanoethyl phosphate

6-(biotinyloxy)hexyl-2-chlorophenyl-2-cyanoethyl phosphate; and

penta(methyl-1-levulinyloxy)dipentaerythrityl-2-chlorophenyl 2-cyanoethyl phosphate;

wherein the linking agent is stable under conditions of polynucleotide synthesis, and wherein said first polynucleotide is attached to the linking agent via the phosphoramidite or phosphate group, and wherein said liking agent and said first polynucleotide together form an adapter molecule;

(b) reacting said adapter molecule with said preselected material; and

(c) ligating said adapter molecule with a second polynucleotide through the 5'"'"' or 3'"'"' end of said second polynucleotide to the 5'"'"' or 3'"'"' end of the first polynucleotide not attached to the adapter molecule.

4. A method for attaching a polynucleotide to a preselected material selected from the group consisting of a solid support, an insoluble matrix, a protein, a small molecule, and a label which comprises:

(a) attaching a first polynucleotide, through its 5'"'"' or 3'"'"' end, to a linking agent having a phosphoramidite or phosphate group, wherein said linking agent is selected from the group consisting of

12-methylcarboxyldodecylmethyl-N,N-diisopropylphosphoramidite;

5-oxohexylmethyl-N,N-diisopropylphosphoramidite;

12-phenyl-12-oxododecylmethyl-N-morpholinophosphoramidite;

12-methyl-11,12-diacetyltridecanediol methyl N-morpholinophosphoramidite;

12-methylcarboxyldodecyl 2-chlorophenyl-2-cyanoethylphosphate; and

penta(methyl-1-levulinyloxy)dipentaerythrityl-2-chlorophenyl 2-cyanoethyl phosphate;

wherein said linking agent is stable under the condition of polynucleotide synthesis, wherein said first polynucleotide is attached to said linking agent via the phosphoramidite or phosphate group, and wherein said linking agent and said first polynucleotide together form an adapter molecule;

(b) ligating said adapter molecule with a second polynucleotide, through the 5'"'"' or 3'"'"' end of said second polynucleotide to the 5'"'"' or 3'"'"'0 end of the first polynucleotide not attached to the adapter molecule, to form a functionalized polynucleotide; and

(c) reacting said functionalized polynucleotide with said preselected material.