# US 2020/0399683 A1 · Composition and Methods For Affinity Directed Enrichment of Rare Species

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- Number: US 2020/0399683 A1
- Document ID: US20200399683A1
- Status: Published application
- Kind: A1
- Application number: 16/975,634
- Filed: 2019-02-26
- Published: 2020-12-24
- Earliest claimed: 2018-02-26
- Assignees: CY Molecular Diagnostics, Inc.
- Applicants: CY Molecular Diagnostics, Inc.
- Inventors: William L. Hanna; Alexander Yum
- Web page: https://patentgrep.com/patent/US20200399683A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6827: for detection of mutation or polymorphism
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Compositions and methods for the selective extraction of rare molecular species from a mixture containing a predominance of related molecular species, and their subsequent release are provided. Compositions include a segregating probe having a target-specific targeting portion, a protecting portion, a scissile or cleavable site or portion, and a capture portion. Complex formation between the targeting portion and the target rare species results in protection of the cleavable site. Such complexes remain intact through a cleavage step directed to the cleavable site or portion of the segregating probe. While complex formation can occur between a segregating probe and a related, but non-target, species the cleavable site is not protected in such complexes. A subsequent capture step directed to the capture portion selectively captures or segregates the target rare species from the general milieu, which can be removed by washing. Subsequent release of the target rare species from the segregating probe provides a solution enriched in the target rare species.

## Claims

### Claim at position 1

1-27. (canceled)

### Claim 28

A method for segregating rare species comprising:
contacting a segregating probe with a sample comprising a mixture of a rare species and a related common species to form a first reaction mixture, wherein the common species is present in at least a 5-fold molar excess over the rare species;
permitting a first complex comprising the segregating probe and the rare species and a plurality of second complexes comprising the segregating probe and the common species to form in the first reaction mixture;
contacting the first reaction mixture with a cleavage reagent to form a second reaction mixture, wherein the cleavage reagent results in cleavage of a scissile bond of the segregating probe of the plurality of second complexes while leaving the scissile bond of the segregating probe of the first complex intact;
contacting the second reaction mixture with a capture species to form a third complex comprising the capture species and the first complex; and
segregating the second complex from the third complex,
wherein the segregating probe comprises a targeting moiety capable of forming a complex with either of the rare species and the common species, a capture tag capable of forming a complex with the capture species, and wherein the scissile bond is interposed between the targeting moiety and the capture tag.

### Claim 29 (depends on 28)

The method of claim 28, wherein the rare species is released from the third complex following segregation.

### Claim 30 (depends on 28)

The method of claim 28, wherein the rare species and the common species are polynucleotides.

### Claim 31 (depends on 28)

The method of claim 28, wherein the rare species is amplified following segregation from the common species.

### Claim 32 (depends on 28)

The method of claim 28, wherein the targeting moiety is selected from the group consisting of a polynucleotide, and antibody, an antibody fragment, and an antibody derivative.

### Claim 33 (depends on 28)

The method of claim 28, wherein the scissile bond is selected from the group consisting of a carbonyl ester or a sulfonamide ester.

### Claim 34 (depends on 28)

The method of claim 28, wherein the capture tag is selected from the group consisting of biotin, iminobiotin, digoxigenin, a polynucleotide, a polypeptide, polyhistidine, an antibody, an antibody fragment, and an antibody derivative.

### Claim 35 (depends on 28)

The method of claim 28, wherein the capture species further comprises a particle, a magnetically responsive particle, a membrane, a well of a microwell plate, an interior surface of a pipette tip or vial, and a filter.

### Claim 36

A segregating probe, comprising:
a targeting moiety configured to form a complex with rare species and a common species;
a capture tag;
a scissile bond interposed between the targeting moiety and the capture tag; and
a protective group,
wherein the protective group is configured to provide a reduced rate of cleavage of the scissile bond in a first complex comprising the segregating probe and the rare species relative to the rated of cleavage of the scissile bond in a second complex comprising the segregating probe and the common species.

### Claim 37 (depends on 36)

The probe of claim 36, wherein the rare species and the common species are polynucleotides.

### Claim 38 (depends on 36)

The probe of claim 36, wherein the targeting moiety is selected from the group consisting of a polynucleotide, and antibody, an antibody fragment, and an antibody derivative.

### Claim 39 (depends on 36)

The probe of claim 36, wherein the scissile bond is selected from the group consisting of a carbonyl ester or a sulfonamide ester.

### Claim 40 (depends on 36)

The probe of claim 36, wherein the capture tag is selected from the group consisting of biotin, iminobiotin, digoxigenin, a polynucleotide, a polypeptide, polyhistidine, an antibody, an antibody fragment, and an antibody derivative, and is selected to form a third complex with a capture species.

### Claim 40

An intermediate for preparation of a segregating probe, comprising:
a reactive group configured to couple to a targeting moiety, wherein the targeting moiety is configured to form a complex with rare species and a common species;
a capture tag;
a scissile bond interposed between the targeting moiety and the capture tag; and
a protective group,
wherein the protective group is configured to provide a reduced rate of cleavage of the scissile bond in a first complex comprising the segregating probe and the rare species relative to the rated of cleavage of the scissile bond in a second complex comprising the segregating probe and the common species.

### Claim 41 (depends on 40)

The intermediate of claim 40, wherein the rare species and the common species are polynucleotides.

### Claim 42 (depends on 40)

The intermediate of claim 40, wherein the targeting moiety is selected from the group consisting of a polynucleotide, and antibody, an antibody fragment, and an antibody derivative.

### Claim 43 (depends on 40)

The intermediate of claim 40, wherein the scissile bond is selected from the group consisting of a carbonyl ester or a sulfonamide ester.

### Claim 44 (depends on 40)

The intermediate of claim 40, wherein the capture tag is selected from the group consisting of biotin, iminobiotin, digoxigenin, a polynucleotide, a polypeptide, polyhistidine, an antibody, an antibody fragment, and an antibody derivative, and is selected to form a third complex with a capture species.