Aptamers specific for biomolecules and methods of making

• US 5,756,291 A
• Filed: 06/07/1995
• Issued: 05/26/1998
• Est. Priority Date: 08/21/1992
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Latham, John, Vermaas, Eric, Leung, Lawrence, Toole, John J., Griffin, Linda, Albrecht, Glenn
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/484,192
• Time in Patent Office: 1,084 Days
• Field of Search: 435/6, 935/77, 935/78, 530/413, 536/23.1
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/6429 Thrombin (3.4.21.5)
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 304/21005 Thrombin (3.4.21.5)

Abstract

A method for identifying oligomer sequences, optionally comprising modified base, which specifically bind target molecules such as serum proteins, kinins, eicosanoids and extracellular proteins is described. The method is used to generate aptamers that bind to serum Factor X, PDGF, FGF, ICAM, VCAM, E-selectin, thrombin, bradykinin, PGF2 and cell surface molecules. The technique involves complexation of the 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 complex is then separated from uncomplexed oligonucleotides and the complexed members of the oligonucleotide mixture are recovered from the separated complex 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. The oligonucleotides can be used for therapeutic and diagnostic purposes and for generating secondary aptamers.

Claims

1. A method to detect the presence or absence of thrombin, which method comprises:

a) contacting a sample suspected of containing thrombin with a single-stranded DNA aptamer coupled to a label under conditions wherein a complex between thrombin and the aptamer is formed; and

b) detecting the presence or absence of said complex indicating the presence or absence of thrombin;

wherein said aptamer comprises the sequence;

##STR17## wherein N is A, T or G.

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

3. The method of claim 1 wherein said aptamer comprises the sequence:

##STR18## wherein;

N is A, T, or G; and

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 said aptamer comprises the sequence:

##STR19##

6. The method of claim 5 wherein N is T (SEQ ID NO:

29).

7. A method to purify thrombin, which method comprises:

a) contacting a sample containing thrombin with a single-stranded DNA aptamer coupled to solid support under conditions wherein thrombin is bound to the aptamer coupled to solid support;

b) washing unbound components of the sample; and

c) recovering purified thrombin from said solid support;

wherein said aptamer comprises the sequence;

##STR20## wherein N is A, T or G.

8. The method of claim 7 wherein N is T.

9. The method of claim 7 wherein said aptamer comprises the sequence:

##STR21## wherein;

N is A, T, or G; and

Z is an integer from 2 to 5.

10. The method of claim 9 wherein Z is 3.

11. The method of claim 10 wherein said aptamer comprises the sequence:

##STR22##

12. The method of claim 11 wherein N is T (SEQ ID NO:

29).