# US 2019/0169689 A1 · Compositions and methods for analyzing nucleic acids associated with an analyte

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- Number: US 2019/0169689 A1
- Document ID: US20190169689A1
- Status: Published application
- Kind: A1
- Application number: 16/324,836
- Filed: 2017-08-11
- Published: 2019-06-06
- Earliest claimed: 2016-08-12
- Assignees: Cdi Laboratories, Inc.
- Applicants: Cdi Laboratories, Inc.
- Inventors: Heng ZHU; Ignacio PINO
- Web page: https://patentgrep.com/patent/US20190169689A1

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6869: Methods for sequencing
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

This disclosure provides compositions and methods for analyzing a nucleic acid associated with an analyte.

## Claims

### Claim 1

A composition comprising:
a. a first probe, wherein the first probe comprises a first tag comprising a polynucleotide comprising a region for attaching to a first end of a nucleic acid; and
b. a second probe, wherein the second probe comprises a second tag comprising a polynucleotide comprising a region for attaching to a second end of the nucleic acid,
wherein the first probe has an affinity to a first binding site on an analyte and the second probe has an affinity to a second binding site on the analyte, wherein the first probe and the second probe are in spatial proximity, and
i. wherein the first probe is associated with a substrate;
ii. wherein the second probe is associated with the substrate;
iii. wherein the first probe is associated with the substrate and wherein the second probe is associated with the substrate;
iv. wherein the first tag is double stranded where associated with the first probe;
v. wherein the second tag is double stranded where associated with the second probe;
vi. wherein the first tag is double stranded where associated with the first probe and wherein the second tag is double stranded where associated with the second probe; or
vii. one of (i), (ii), or (iii) and one of (iv), (v) or (vi).

### Claim 2 (depends on 1)

The composition of claim 1, wherein the first probe is associated with a solid substrate.

### Claim 3 (depends on 1, 2)

The composition of claim 1 or 2, wherein the second probe is associated with the solid substrate.

### Claim 4 (depends on 1, 2)

The composition of any one of claim 1, 2, or 3, wherein the solid substrate is planar.

### Claim 5 (depends on 1, 2, 3, 4)

The composition of any one of claims 1-4, wherein the solid substrate is an array.

### Claim 6 (depends on 1, 2)

The composition of any one of claim 1, 2, or 3, wherein the solid substrate is spherical.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the spherical solid substrate is a bead.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the bead is a Sepharose bead.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7)

The composition of any one of claims 1-7, wherein at least a portion of the solid substrate is coated.

### Claim 10 (depends on 9)

The composition of claim 9, wherein at least a portion of the solid substrate is contacted with at least one of a polymer or a first binding partner which has an affinity for a second binding partner.

### Claim 11 (depends on 10)

The composition of claim 10 comprising the polymer, wherein the polymer is selected from the group consisting of polyethylene glycol, polymethacrylate, polymethylmethacrylate, polyethylenimine, polyvinyl alcohol, polyvinyl acetate, polystyrene, polyglutaraldehyde, polyacrylamide, agarose, chitosan, alginate, and a combination thereof.

### Claim 12 (depends on 10)

The composition of claim 10 comprising the first binding partner which has an affinity for the second binding partner, wherein the first binding partner is selected from the group consisting of immunoglobulin-binding protein, calmodulin, glutathione, glutathione S-transferase (GST), streptavidin, avidin, maltose-binding protein, a His tag, and a combination thereof.

### Claim 13 (depends on 10)

The composition of claim 10 comprising the first binding partner which has an affinity for the second binding partner, wherein the second binding partner is selected from the group consisting of immunoglobulin-binding protein, calmodulin, glutathione, glutathione S-transferase (GST), streptavidin, avidin, maltose-binding protein, a His tag, and a combination thereof.

### Claim 14 (depends on 12, 13)

The composition of claim 12 or 13, comprising the immunoglobulin-binding protein wherein the immunoglobulin-binding protein is Protein A or Protein G.

### Claim 15 (depends on 11)

The composition of claim 11, wherein each of the first probe and the second probe comprise at least one of a binding partner of the polymer or the second binding partner.

### Claim 16 (depends on 15)

The composition of claim 15 comprising the second binding partner, wherein the first binding partner is GST and the first probe and the second probe comprise glutathione.

### Claim 17

The composition of any one of the above claims, wherein the solid substrate is magnetic.

### Claim 18 (depends on 17)

The composition of claim 17, wherein the magnetic solid substrate comprises magnetite, maghemitite, FePt, SrFe, iron, cobalt, nickel, chromium dioxide, ferrites, or a mixture thereof.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The composition of any one of claims 1-16, wherein the solid substrate is nonmagnetic.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the first probe comprises a first antibody or a fragment thereof, and wherein each first antibody or the fragment thereof comprises at least one of a binding partner of the polymer or the second binding partner.

### Claim 21 (depends on 1, 20)

The composition of claim 1 or 20, wherein the second probe comprises a second antibody or a fragment thereof, and wherein each second antibody or the fragment thereof comprises at least one of a binding partner of the polymer or the second binding partner.

### Claim 22 (depends on 20, 21)

The composition of claim 20 or 21, wherein the first antibody or the second antibody is a monoclonal antibody, a recombinant antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a fragment thereof.

### Claim 23 (depends on 20, 21)

The composition of claim 20 or 21, wherein the first antibody or the second antibody is isolated or purified from a hybridoma.

### Claim 24 (depends on 20, 21, 22, 23)

The composition of any one of claims 20-23, wherein the first antibody or the fragment thereof is conjugated with the first tag and the second antibody or the fragment thereof is conjugated with the second tag.

### Claim 25 (depends on 1)

The composition of claim 1, wherein the first tag is double stranded.

### Claim 26

The composition of any one of the above claims, wherein the second tag is double stranded.

### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)

The composition of any one of claims 1-25, wherein the second tag is single stranded.

### Claim 28

The composition of any one of the above claims, wherein the first tag comprises a first cleavage site.

### Claim 29

The composition of any one of the above claims, wherein the second tag comprises a second cleavage site.

### Claim 30 (depends on 28, 29)

The composition of claim 28 or 29, wherein the first cleavage site and the second cleavage site are endonuclease recognition sites.

### Claim 31 (depends on 30)

The composition of claim 30, wherein the endonuclease sites comprises type II endonuclease recognition sites.

### Claim 32 (depends on 31)

The composition of claim 31, wherein the type II endonuclease recognition sites are BsaI recognition sites.

### Claim 33

The composition of any one of the above claims, wherein the first tag comprises a first barcode.

### Claim 34

The composition of any one of the above claims, wherein the second tag comprises a second barcode.

### Claim 35 (depends on 33)

The composition of claim 33, wherein the first barcode comprises about 1 to 50 nucleotides.

### Claim 36 (depends on 33, 34, 35)

The composition of claims 33, 34 or 35, wherein the second barcode comprises about 1 to 50 nucleotides.

### Claim 37 (depends on 33)

The composition of claim 33, wherein the first tag comprises a first primer binding site, and the second tag comprises a second primer binding site.

### Claim 38 (depends on 33, 34, 35, 36, 37)

The composition of any one of claims 33-37, wherein the first probe is uniquely identifiable by the first barcode.

### Claim 39 (depends on 33, 34, 35, 36, 37, 38)

The composition of any one of claims 33-38, wherein the second probe is uniquely identifiable by the second barcode.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The composition of any one of claims 1-39, wherein the first polynucleotide and the second polynucleotide are DNA.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The composition of any one of claims 1-39, wherein the first polynucleotide and the second polynucleotide are RNA.

### Claim 42 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The composition of any one of claims 1-39, wherein the first polynucleotide and the second polynucleotide are a hybrid of DNA and RNA.

### Claim 43

The composition of any of the above claims, wherein the analyte comprise a first biological molecule.

### Claim 44 (depends on 43)

The composition of claim 43, wherein the first biological molecule is a protein, a carbohydrate, a lipid, or a nucleic acid.

### Claim 45 (depends on 44)

The composition of claim 44, wherein the analyte comprises a first protein.

### Claim 46 (depends on 45)

The composition of claim 45, wherein the first protein comprises a first modified residue and a second modified residue.

### Claim 47 (depends on 46)

The composition of claim 46, wherein the first probe binds to an antigen comprising the first modified residue and the second probe binds to an antigen comprising the second modified residue.

### Claim 48 (depends on 46, 47)

The composition of claim 46 or 47, wherein modification on the first modified residue is methylation, phosphorylation, acetylation, ubiquitylation, sumoylation, or a combination thereof.

### Claim 49 (depends on 46, 47, 48)

The composition of any one of claims 46-48, wherein modification on the second modified residue is methylation, phosphorylation, acetylation, ubiquitylation, sumoylation, or a combination thereof.

### Claim 50 (depends on 46, 47, 48, 49)

The composition of any one of claims 46-49, wherein the first protein is a histone.

### Claim 51 (depends on 50)

The composition of claim 50, wherein the histone is modified.

### Claim 52 (depends on 51)

The composition of claim 51, wherein the modification is methylation, acetylation, or a combination thereof.

### Claim 53 (depends on 50, 51)

The composition of claim 50 or 51, wherein the histone is histone 3.

### Claim 54 (depends on 50, 51, 52, 53)

The composition of any one of claims 50-53, wherein the histone is modified at a lysine residue.

### Claim 55

The composition of any one of the above claims, wherein the analyte further comprises a second protein.

### Claim 56 (depends on 55)

The composition of claim 55, wherein the first protein or the second protein comprises a transcription factor.

### Claim 57 (depends on 55, 56)

The composition of claim 55 or 56, wherein the first protein and the second protein form a dimer.

### Claim 58 (depends on 55, 56, 57)

The composition of any one of claims 55-57, wherein the first protein comprises the first binding site and the second protein comprises the second binding site.

### Claim 59

The composition of any one of the above claims, wherein the analyte is associated with a nucleic acid.

### Claim 60 (depends on 59)

The composition of claim 59, wherein the nucleic acid comprises genomic DNA.

### Claim 61 (depends on 59)

The composition of claim 59, wherein the nucleic acid is intracellular or extracellular.

### Claim 62 (depends on 59)

The method of claim 59, wherein the nucleic acid is RNA, DNA, or a hybrid thereof.

### Claim 63

The composition of any one of the above claims, wherein the composition is in the form of an array.

### Claim 64

A method comprising:
contacting a sample comprising a nucleic acid associated with an analyte with
a. a first probe, wherein the first probe comprises a first tag comprising a polynucleotide comprising a region for attaching to a first end of a nucleic acid; and
b. a second probe, wherein the second probe comprises a second tag comprising a polynucleotide comprising a region for attaching to a second end of the nucleic acid,
wherein the first probe has an affinity to a first binding site on the analyte and the second probe has an affinity to a second binding site on the analyte, wherein the first probe and the second probe are in spatial proximity, and
i. wherein the first probe is associated with a substrate;
ii. wherein the second probe is associated with the substrate;
iii. wherein the first probe is associated with the substrate and wherein the second probe is associated with the substrate;
iv. wherein the first tag is double stranded where associated with the first probe;
v. wherein the second tag is double stranded where associated with the second probe;
vi. wherein the first tag is double stranded where associated with the first probe and wherein the second tag is double stranded where associated with the second probe; or
vii. one of (i), (ii), or (iii) and one of (iv), (v) or (vi).

### Claim 65

A method comprising:
a. extracting an analyte with a nucleic acid associated with the analyte from a sample by contacting the sample with an extraction complex comprising an extraction moiety and an oligonucleotide, wherein the extraction complex binds to the nucleic acid; and
b. contacting the extracted analyte with:
i. a first probe that has an affinity to a first binding site on the analyte, and
ii. a second probe that has an affinity to a second binding site on the analyte, wherein the first probe comprises a first tag comprising a first polynucleotide comprising a region for attaching to a first end of the nucleic acid, and the second probe comprises a second tag comprising a second polynucleotide comprising a region for attaching to a second end of the nucleic acid, and wherein the first probe and the second probe are in spatial proximity.

### Claim 66 (depends on 65)

A method of claim 65, further comprising calculating, with one or more computer processors, a first value of at least one parameter, corresponding to a transcriptional efficiency of at least a portion of the nucleic acid associated with the analyte, and wherein the transcriptional efficiency is correlated to a presence of at least one of the first binding site or the second binding site on the analyte.

### Claim 67 (depends on 66)

A method of claim 66, further comprising comparing, with the use of one or more computer processors, the first value of the at least one parameter to a reference value.

### Claim 68 (depends on 67)

A method of claim 67, further comprising identifying, with the use of one or more computer processors, a disease in the subject if the first value of the first parameter exceeds the reference value.

### Claim 69 (depends on 64, 65, 66, 67, 68)

The method of any one of claims 64-68, wherein the sample is a biological sample.

### Claim 70 (depends on 69)

The method of claim 69, wherein the biological sample is selected from the group consisting of amniotic fluid, blood plasma, blood serum, breast milk, cells, cancer cells, tumor cells, cerebrospinal fluid, saliva, semen, synovial fluid, tears, tissue, cancer tissue, tumor tissue, urine, white blood cells, whole blood, and any fraction thereof.

### Claim 71

A method comprising:
a. associating a substrate to a first probe and a second probe, wherein the first probe comprises a first tag comprising a first polynucleotide and the second probe comprises a second tag comprising a second polynucleotide, wherein the first probe has an affinity to a first binding site on an analyte in a sample, and the second probe has an affinity to a second binding site on the analyte, wherein the first tag comprises a region for attaching to a first end of a nucleic acid associated with the analyte, and the second tag comprises a region for attaching to a second end of the nucleic acid associated with the analyte.

### Claim 72 (depends on 64, 65, 66, 67, 68, 69, 70, 71)

The method of any one of claims 64-71, wherein the nucleic acid is an intracellular nucleic acid.

### Claim 73 (depends on 64, 65, 66, 67, 68, 69, 70, 71)

The method of any one of claims 64-71, wherein the nucleic acid is an extracellular nucleic acid.

### Claim 74 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)

The method of any one of claims 64-73, wherein the nucleic acid is DNA.

### Claim 75 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)

The method of any one of claims 64-73, wherein the nucleic acid is RNA.

### Claim 76 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)

The method of any one of claims 64-73, wherein the nucleic acid is a hybrid of DNA and RNA.

### Claim 77 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76)

The method of any one of claims 64-76, further comprising modifying the nucleic acid, wherein the modifying comprises generating a single stranded overhang at the first end of the nucleic acid or at the second end of the nucleic acid.

### Claim 78 (depends on 64, 71)

The method of claim 64 or 71, further comprising extracting the nucleic acid associated with the analyte from the sample by contacting the sample with an extraction complex comprising an extraction moiety and an oligonucleotide, wherein the extraction complex binds to the nucleic acid.

### Claim 79 (depends on 65, 66, 67, 68, 78)

The method of any one of claims 65-68 or 78, wherein the extraction moiety is biotin or a fragment thereof.

### Claim 80 (depends on 65, 66, 67, 68, 78, 79)

The method of any one of claims 65-68 or 78-79, wherein the extraction complex comprises a polynucleotide linker.

### Claim 81 (depends on 65, 66, 67, 68, 78, 79, 80)

The method of any one of claims 65-68 or 78-80, wherein the oligonucleotide binds to the nucleic acid associated with the analyte.

### Claim 82 (depends on 65, 66, 67, 68, 78, 79, 80, 81)

The method of any one of claims 65-68 or 78-81, further comprising dissociating the nucleic acid associated with the analyte from the extraction complex.

### Claim 83 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)

The method of any one of claims 64-82, wherein at least one of the first probe binds to the first binding site on the analyte or the second probe binds to the second binding site on the analyte.

### Claim 84 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83)

The method of any one of claims 64-83, further comprising attaching the first tag to the first end of the nucleic acid associated with the analyte and the second tag to the second end of the nucleic acid associated with the analyte.

### Claim 85 (depends on 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)

The method of any one of claims 64-84, further comprising analyzing the nucleic acid, wherein analyzing the nucleic acid comprises at least one of amplifying the nucleic acid or sequencing the nucleic acid.

### Claim 86 (depends on 85)

The method of claim 85, wherein the sequencing comprises multiplex sequencing.

### Claim 87 (depends on 85)

The method of claim 85, wherein the amplifying comprises polymerase chain reaction.

### Claim 88 (depends on 64, 71)

The method of any one of claim 64 or 71, wherein the substrate is an array.

### Claim 89 (depends on 64, 71)

The method of any one of claim 64 or 71, wherein the substrate is a bead.

### Claim 90 (depends on 89)

The method of claim 89, wherein the bead is a Sepharose bead.

### Claim 91 (depends on 64, 65, 66, 67, 68, 69, 70)

The method of any one of claims 64-70, wherein the method is at least partially performed as a liquid phase assay.