Aptamers and methods for their in vitro selection and uses thereof

• US 20090075834A1
• Filed: 02/06/2008
• Published: 03/19/2009
• 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: Murphy, Michael B., Doyle, Sharon A.
• Granted Patent: US 8,105,982 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 having high affinity to a target molecule, comprising:

(a) providing a target molecule with a polyhistidine affinity tag for magnetic beads;

(b) binding the target molecule to magnetic beads then contacting the target molecule with a library of degenerate aptamer sequences to allow binding of aptamer sequences to the target molecule then forming bead-target-aptamer sequence complexes, wherein each of said degenerate sequences is flanked by fixed sequences which permit ligation-dependent cloning;

(c) separating bead-target-aptamer sequence complexes from the library of non-binding aptamer sequences;

then

(d) separating target-bound aptamer sequences from said magnetic beads to form a pool of binding aptamer sequences;

(e) amplifying the binding aptamer sequences; and

,

(f) iteratively repeating steps (b) through (d) a sufficient number of times to result in identification of at least one aptamer sequence having high affinity for the target molecule.

2. The method of claim 1 further comprising:

subcloning the high affinity aptamer sequence into a plasmid and transforming E. coli;

isolating said plasmid containing said aptamer sequence from transformed E. coli; and

amplifying and sequencing said cloned aptamer sequence.

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

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

35. The method of claim 1, wherein the target molecule is a polyhistidine tag and the aptamer obtained by the method of claim 1 comprising a degenerate consensus sequence SEQ ID NO:

21, wherein said sequence exhibits binding activity to polyhistidine tags.

36. The method of claim 35, wherein the aptamer comprising a degenerate sequence SEQ ID NO:

14, wherein said sequence exhibits binding activity to polyhistidine tags.

37. The method of claim 35, wherein the aptamer comprising a degenerate sequence SEQ ID NO:

15, wherein said sequence exhibits binding activity to polyhistidine tags.

38. The method of claim 35, wherein the aptamer comprising a degenerate sequence SEQ ID NO:

16, wherein said sequence exhibits binding activity to polyhistidine tags.

3-4. -4. (canceled)

7-34. -34. (canceled)