Sperm cell separation methods and compositions containing aptamers or nucleic acid sequences for use therein

• US 8,138,319 B2
• Filed: 02/27/2009
• Issued: 03/20/2012
• Est. Priority Date: 02/24/2005
• Status: Active Grant

• Current Assignee: Biocern Inc.
• Original Assignee: Biocern Inc.
• Inventors: Krylov, Sergey, Musheev, Michael, Oksenberg, David
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/394,993
• Publication Number:
  US 20090306461A1
• Time in Patent Office: 1,117 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 2320/10 in screening processes
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6879 for sex determination

Abstract

The present invention provides an aptamer or pool of aptamers (nucleic acid sequences) that bind(s) to a target molecule on the surface, accessible from the surface or inside of a mammalian sperm cell and a method for producing the aptamers. The method comprises contacting a collection of different nucleic acid molecules with the target molecule under conditions favorable for binding between at least one of the nucleic acid molecules and the target molecule, to form at least one complex comprising the nucleic acid molecule bound to the target molecule, wherein each of the nucleic acid molecules comprises at least one segment of randomized nucleotide sequences. The complexes are then separated from the unbound nucleic acid molecules and unbound target molecules, and the bound nucleic acid molecule is recovered from the separated complex. The aptamers are used to separate sperm cells based upon sperm cell qualities, such as whether the cells contain X chromosomes or Y chromosomes.

Claims

1. An isolated non-naturally occurring nucleic acid sequence that binds to a target molecule accessible from the surface of a mammalian sperm cell comprising

a nucleotide sequence selected from the group consisting of SEQ ID NOS:

3-35.

2. A composition comprising at least one nucleic acid sequence of claim 1.

3. A method of separating a mixture of mammalian sperm cells containing at least one target molecule comprising:

contacting the mammalian sperm cells with the composition of claim 2 for a period of time sufficient to allow binding of the nucleic acid sequence to the sperm cells;

separating the sperm cells into two populations of sperm cells, one population comprising X sperm cells and the other comprising Y sperm cells based on the ability of the at least one nucleic acid sequence to bind to a target molecule accessible from the surface of a sperm cell; and

optionally;

recovering the separated sperm cells.

4. The method of claim 3, wherein the nucleic acid sequence

binds preferentially to the X sperm cells and a population of predominantly X sperm cells are recovered.

5. The method of claim 3, wherein the mammalian sperm cells are cattle sperm cells or human sperm cells.

6. The method of claim 3, wherein at least one of the sperm cell populations comprises sperm cells unbound to the nucleic acid sequence or sperm cells bound to the nucleic acid sequence.

7. The method of claim 3, wherein the composition comprises two or more different nucleic acid sequence.

8. The method of claim 3, wherein the nucleic acid sequence is bound to a non-magnetic bead or a magnetic bead.

9. The separated sperm cells produced by the method of claim 3 comprising sperm cells bound to the at least one nucleic acid sequence.

10. The separated sperm cells of claim 9, wherein said sperm cells are cattle sperm cells.

11. The separated sperm cells of claim 9, wherein said sperm cells are human sperm cells.

12. A method for separating X sperm cells and Y sperm cells comprising the steps of:

(a) contacting a mixed population of X and Y sperm cells with a nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS;

3-35,which nucleic acid sequence preferentially binds to the X sperm cells to form bound nucleic acid-X sperm cell complexes;

(b) separating the X sperm cells from the original mixed sperm cell population leaving a Y containing rich sperm cell population;

(c) optionally releasing the bound X sperm cells from the nucleic acid sequence-X sperm cell complexes; and

(d) recovering the X sperm cells or the Y sperm cells.

13. The method of claim 12, wherein said Y sperm cells are recovered from step (b).