Aptamers and methods for their in vitro selection and uses thereof

• US 8,105,982 B2
• Filed: 02/06/2008
• Issued: 01/31/2012
• Est. Priority Date: 09/04/2003
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Doyle, Sharon A., Murphy, Michael B.
• Primary Examiner(s): Steele, Amber D.
• Application Number:
  US12/027,239
• Publication Number:
  US 20090075834A1
• Time in Patent Office: 1,455 Days
• Field of Search: 536/23.1, 506/4, 506/9
• US Class Current: 506/4
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Claims

1. A method for obtaining an aptamer sequence having high affinity to a target molecule, comprising:

(a) providing a target molecule with a polyhistidine tag, said polyhistidine tag having a binding affinity to magnetic beads;

(b) binding the target molecule to magnetic beads via a 6His aptamer, wherein the magnetic beads are coated with the 6His aptamer comprising the aptamer sequence of SEQ ID NOS;

14, 15, 16 and/or 21;

(c) providing a library of degenerate aptamer sequences, wherein each of said degenerate aptamer sequences is flanked by fixed sequences which permit ligation-dependent cloning;

(d) contacting the magnetic bead-bound target molecule with said library of degenerate aptamer sequences to allow binding of said degenerate aptamer sequences to the target molecule with high affinity, wherein said high affinity has a dissociation constant of at least 10<sup>−

4</sup>M;

(e) forming magnetic bead-bound target-aptamer sequence complexes upon binding of said degenerate aptamer sequences to the target molecule with high affinity;

(f) separating said magnetic bead-bound target-aptamer sequence complexes from the library of non-binding degenerate aptamer sequences;

(g) separating target-bound degenerate aptamer sequences from said magnetic bead-bound target aptamer sequence complexes to form a pool of high affinity binding aptamer sequences;

(h) amplifying the high affinity binding aptamer sequences; and

(i) iteratively repeating steps (b) through (h) a sufficient number of times to result in identification of at least one high affinity binding aptamer sequence having high affinity for the target molecule, thus obtaining an aptamer sequence having high affinity to said target molecule.

2. The method of claim 1 further comprising:

(j) subcloning the high affinity binding aptamer sequence into a plasmid and transforming E. coli;

(k) isolating said plasmid containing said high affinity binding aptamer sequence from transformed E. coli; and

(l) amplifying and sequencing said cloned high affinity binding aptamer sequence.

3. The method of claim 1 wherein said flanking fixed sequences are primer sequences.

4. The method of claim 2 wherein said amplification is by rolling circle amplification.

5. The method of claim 1, wherein the 6His aptamer consists of SEQ ID NO:

21 and wherein SEQ ID NO;

21 exhibits binding activity to polyhistidine tags.

6. The method of claim 1, wherein the 6His aptamer consists of SEQ ID NO:

14 and wherein SEQ ID NO;

14 exhibits binding activity to polyhistidine tags.

7. The method of claim 1, wherein the 6His aptamer consists of SEQ ID NO:

15 and wherein SEQ ID NO;

15 exhibits binding activity to polyhistidine tags.

8. The method of claim 1, wherein the 6His aptamer consists of SEQ ID NO:

16 and wherein SEQ ID NO;

16 exhibits binding activity to polyhistidine tags.