Method for identifying an oligonucleotide aptamer specific for a target

• US 5,582,981 A
• Filed: 04/28/1994
• Issued: 12/10/1996
• Est. Priority Date: 08/14/1991
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Latham, John, Toole, John J., Griffin, Linda C., Bock, Louis C.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/234,613
• Time in Patent Office: 957 Days
• Field of Search: 435/6, 435/91.2, 935/77
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: G01N 2333/974 Thrombin
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

A method for identifying oligomer sequences which specifically bind target biomolecules is described. The technique involves complexation of the support-bound target molecule with a mixture of oligonucleotides containing random sequences and sequences which serve as primer for PCR under conditions wherein a complex is formed with the specifically binding sequences, but not with the other members of the oligonucleotide mixture. The target-oligonucleotide complexes are then separated from both the support and the uncomplexed oligonucleotides and the complexed members of the oligonucleotide mixture are recovered from the separated complex and subsequently amplified using the polymerase chain reaction. The recovered oligonucleotides may be sequenced, and successive rounds of selection using complexation, separation, amplification and recovery can be employed.

Claims

1. A method for identifying an aptamer which specifically binds to thrombin, wherein the aptamer comprises the sequence 5'"'"' GGNTGG 3'"'"' wherein N is A, T Or G which method comprises:

(a) incubating thrombin reversibly coupled to a support with a randomized mixture of single-stranded DNA oligonucleotide sequences under conditions wherein the member of the mixture to form a support-bound oligonucleotide-thrombin complex;

(b) decoupling and recovering support-bound oligonucleotide-thrombin complex to form a free oligonucleotide-thrombin complex;

(c) recovering free oligonucleotide from the free oligonucleotide-thrombin complex and recovering and amplifying the free oligonucleotide; and

(d) optionally determining the sequence of the recovered oligonucleotide.

2. The method of claim 1 wherein N is T.

3. The method of claim 1 wherein the aptamer comprises the sequence 5'"'"' GGNTGG(N)_Z GGNTGG 3'"'"' wherein Z is an integer from 2 to 5.

4. The method of claim 3 wherein Z is 3.

5. The method of claim 4 wherein the aptamer comprises the sequence 5'"'"' GGNTGGTGTGGNTGG 3'"'"'.

6. The method of claim 5 wherein N is T.

7. The method of claim 1 wherein the support is a lectin column.

8. The method of claim 1 wherein the support is a solid support.

9. The method of claim 1 wherein the solid support comprises agarose or sepharose.

10. The method of claim 3 wherein the support is a lectin column.

11. The method of claim 3 wherein the support is a solid support.

12. The method of claim 3 wherein the solid support comprises agarose or sepharose.