# US 7,745,607 B2 · Aptamer selection method

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- Number: US 7,745,607 B2
- Document ID: US7745607B2
- Status: Granted
- Kind: B2
- Application number: 10/551,452
- Filed: 2004-03-31
- Published: 2007-10-18
- Granted: 2010-06-29
- Earliest claimed: 2003-03-31
- Assignees: McMaster University
- Inventors: Yingfu Li; Razvan Nutiu
- Web page: https://patentgrep.com/patent/US7745607B2

## CPC

- C12N 15/1048: {SELEX}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention provides a method for the selection of aptamer sequences and aptamers identified using the method. The method comprises immobilizing a library oligonucleotide by forming a duplex with an antisense oligonucleotide, reating the duplex molecule with a target and collecting oligonucleotide molecules which dissociate from the duplex structure.

## Claims

### Claim 1

A method for selecting aptamer capable of binding to a target, said method comprising the steps of: i) interacting antisense oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and being adapted to be attached to a solid support with an oligonucleotide library containing multiple oligonucleotides, each of which includes an antisense binding domain having a sequence complementary to SEQ ID NO: 1 and a random nucleotide domain, wherein the antisense oligonucleotide and the oligonucleotides in the library form double stranded duplexes, ii) immobilizing the duplexes on a solid support; iii) incubating the duplexes in the presence of the target; and iv) collecting library oligonucleotides that dissociate from the duplexes and bind to the target.

### Claim 2 (depends on 1)

The method of claim 1, further comprising amplifying the library oligonucleotides collected at step iv) to provide an amplified population.

### Claim 3 (depends on 2)

The method of claim 2 further comprising the step of sequencing clones derived from the amplified population.

### Claim 4

A method for the selection of an aptamer specific for a target, said method comprising the steps of: i) providing an oligonucleotide library containing multiple oligonucleotides, each of which includes an antisense binding domain having a sequence complementary to SEQ ID NO: 1, at least one random sequence domain, a 3′ primer binding domain and a 5′ primer binding domain; ii) hybridizing the oligonucleotides in the library to a biotinylated antisense oligonucleotide having the nucleotide sequence of SEQ ID NO:1 to form duplex molecules on beads; iii) incubating the beads of ii) with the target; iv) collecting oligonucleotides which have bound to the target; and v) amplifying the collected oligonucleotides.

### Claim 5

An aptamer selection system comprising an antisense oligonucleotide having the nucleotide sequence of SEQ ID NO:1, and an oligonucleotide library containing multiple oligonucleotides, each of which includes an antisense binding domain having a sequence complementary to SEQ ID NO:1 and at least one random nucleotide domain, wherein said antisense oligonucleotide is adapted to be attached to a solid support.

### Claim 6 (depends on 5)

The aptamer selection system according to claim 5, wherein each of the oligonucleotides in the library further comprises a first primer binding domain at the 5′ end and a second primer binding domain at the 3′ end.

### Claim 7 (depends on 6)

The aptamer selection system according to claim 6, further comprising a first primer capable of binding to the first primer binding domain and a second primer capable of binding to the second primer binding domain.

### Claim 8 (depends on 5)

The aptamer selection system according to claim 5, wherein each of the oligonucleotides in the library comprises two random domains flanking the antisense binding domain.

### Claim 9 (depends on 5)

The aptamer selection system according to claim 5, wherein the antisense oligonucleotide is biotinylated.

### Claim 10 (depends on 9)

The aptamer selection system according to claim 9, further comprising avidin agarose beads.

### Claim 11

An aptamer selection system comprising an antisense oligonucleotide comprising SEQ ID. NO. 1, an oligonucleotide library containing multiple oligonucleotides each of which includes SEQ ID. NO. 2, a P1 oligonucleotide comprising SEQ ID. NO. 3, a P2 oligonucleotide comprising SEQ ID NO. NO. 4, and a P3 oligonucleotide comprising SEQ ID. NO. 5.

### Claim 12 (depends on 6)

The aptamer selection system according to claim 6, wherein each of the oligonucleotides in the library comprises two random domains flanking the antisense binding domain.

### Claim 13 (depends on 7)

The aptamer selection system according to claim 7, wherein each of the oligonucleotides in the library comprises two random domains flanking the antisense binding domain.

### Claim 14 (depends on 6)

The aptamer selection system according to claim 6, wherein the antisense oligonucleotide is biotinlylated.

### Claim 15 (depends on 7)

The aptamer selection system according to claim 7, wherein the antisense oligonucleotide is biotinlylated.

### Claim 16 (depends on 8)

The aptamer selection system according to claim 8, wherein the antisense oligonucleotide is biotinlylated.