# US 2021/0239692 A1 · Aptamer-Based Multiplexed Assays

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- Number: US 2021/0239692 A1
- Document ID: US20210239692A1
- Status: Published application
- Kind: A1
- Application number: 17/217,006
- Filed: 2021-03-30
- Published: 2021-08-05
- Earliest claimed: 2012-06-07
- Assignees: SomaLogic, Inc.
- Applicants: SomaLogic, Inc.
- Inventors: Glenn Sanders; Stephan Kraemer; Evaldas Katilius; EDGAR OLIVAS
- Web page: https://patentgrep.com/patent/US20210239692A1

## CPC

- G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding}
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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 disclosure describes methods, devices, reagents, and kits for the detection of one or more target molecules that may be present in a test sample. The described methods, devices, kits, and reagents facilitate the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer) where the aptamer-aptamer interactions are significantly reduced or eliminated while maintaining the aptamer-target interaction.

## Claims

### Claim 1

A method for detecting a target molecule that may be present in a test sample comprising:
exposing an aptamer having specific affinity for the target molecule to a first solid support, wherein the aptamer comprises a first tag and the first solid support comprises a first capture element, and wherein the first tag has affinity for the first capture element;
allowing the first tag to interact with the first capture element to immobilize the aptamer on the first solid support;
washing the first solid support with one or more solutions that dissociate aggregated aptamers;
contacting the immobilized aptamer with a test sample, wherein an aptamer-target affinity complex is formed if the target molecule is present in the test sample;
removing one or more components not immobilized on the first solid support;
attaching a second tag to the target molecule in the aptamer-target affinity complex, wherein the second tag has an affinity to a second capture element;
releasing the aptamer-target affinity complex from said first solid support;
exposing the released aptamer-target affinity complex to a second solid support comprising a second capture element and allowing the second tag to interact with said second capture element to immobilize the aptamer-target affinity complex on the second solid support;
removing one or more components not immobilized on the second solid support eluting the aptamer from the second solid support with one or more buffered solutions comprising a chaotropic salt;
detecting the presence of said target molecule in the test sample by detecting the aptamer of said aptamer-target affinity complex; and
wherein the target molecule is a protein.

### Claim 2 (depends on 1)

The method of claim 1 further comprising quantifying the aptamer.

### Claim 3 (depends on 1)

The method of claim 1 further comprising detecting the aptamer by hybridizing the aptamer to a third solid support, wherein the third solid support comprises a plurality of addressable features and wherein at least one of said features comprises at least one capture element disposed thereon that is complementary to any sequence contained within the aptamer.

### Claim 4 (depends on 1)

The method of claim 1, wherein the aptamer is detected and optionally quantified by a method selected from the group consisting of Q-PCR, MS, next-generation sequencing and hybridization.

### Claim 5 (depends on 4)

The method of claim 4, wherein said Q-PCR is performed by a method selected from the group consisting of TaqMan® PCR, an intercalating fluorescent dye during the PCR process, or a molecular beacon during the PCR process.

### Claim 6 (depends on 1)

The method of claim 1, wherein the pH of the one or more solutions is about 11.

### Claim 7 (depends on 1)

The method of claim 1, wherein the pH of the one or more buffered solutions is neutral.

### Claim 8 (depends on 1)

The method of claim 1, wherein the chaotropic salt disrupts aptamer-target interactions, thereby dissociating the aptamer from the immobilized target.

### Claim 9 (depends on 1)

The method of claim 1, wherein said chaotropic salt is selected from the group consisting of sodium perchlorate, lithium chloride, magnesium chloride and sodium chloride.

### Claim 10 (depends on 1)

The method of claim 1, wherein the one or more of the buffered solutions comprises an organic solvent.

### Claim 11 (depends on 1)

The method of claim 1, wherein the organic solvent is glycerol.

### Claim 12 (depends on 1)

The method of claim 1, wherein the aptamer is a single-stranded nucleic acid or a double-stranded nucleic acid.

### Claim 13 (depends on 1)

The method of claim 1, wherein the aptamer comprises DNA, RNA or both DNA and RNA.

### Claim 14 (depends on 1)

The method of claim 1, wherein the aptamer-target affinity complex has a rate of dissociation greater than or equal (≥) to 30 minutes.

### Claim 15 (depends on 1)

The method of claim 1, wherein the rate of dissociation of the aptamer-target affinity complex (t1/2) is selected from the group consisting of ≥30 minutes, ≥60 minutes, ≥90 minutes, ≥120 minutes, ≥150 minutes, ≥180 minutes, ≥210 minutes, and ≥240 minutes.

### Claim 16

The method of 1, wherein the aptamer comprises a detectable moiety is selected from the group consisting of a dye, a quantum dot, a radiolabel, an electrochemical functional group, and an enzyme plus a detectable enzyme substrate.

### Claim 17 (depends on 16)

The method of claim 16, wherein the dye is a fluorescent dye.

### Claim 18 (depends on 1)

The method of claim 1, wherein the aptamer comprises at least one C-5 modified nucleotide.

### Claim 19 (depends on 1)

The method of claim 1, wherein the aptamer comprises at least one chemical modification comprising a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

### Claim 20 (depends on 19)

The method of claim 19, wherein the chemical modification is independently selected from the group consisting of a 2′-position sugar modification, a 2′-amino (2′-NH2), a 2′-fluoro (2′-F), a 2′-O-methyl (2′-OMe), a 5-position pyrimidine modification, an 8-position purine modification, a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, methylation, a 3′ cap, and a 5′ cap.

### Claim 21 (depends on 1)

The method claim 1, wherein the test sample is selected from the group consisting of blood, whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue, a tissue extract, a tissue biopsy, and cerebrospinal fluid.

### Claim 22 (depends on 1)

The method of claim 1, wherein the first tag and the second tag each comprises at least one component independently selected from the group consisting of a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affibody, a cell receptor, a ligand, a lipid, biotin, avidin, streptavidin, Extravidin, neutravidin, Traptavidin, a metal, histidine, and any portion of any of these structures.

### Claim 23 (depends on 1)

The method of claim 1, wherein said first capture element and said second capture element each comprises at least one component independently selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an affibody, an antibody mimic, a cell receptor, a ligand, a lipid, biotin, avidin, streptavidin, Extravidin, neutravidin, Traptavidin, a metal, histidine, and any portion of any of these structures.

### Claim 24 (depends on 1)

The method of claim 1, wherein the first tag comprises a releasable moiety.

### Claim 25 (depends on 24)

The method of claim 24, wherein the releasable moiety comprises a photocleavable moiety.

### Claim 26 (depends on 1)

The method of claim 1, wherein said first solid support and second solid support each is independently selected from the group consisting of a polymer bead, an agarose bead, a polystyrene bead, an acrylamide bead, a solid core bead, a porous bead, a paramagnetic bead, glass bead, controlled pore bead, a microtitre well, a cyclo-olefin copolymer substrate, a membrane, a plastic substrate, nylon, a Langmuir-Blodgett film, glass, a germanium substrate, a silicon substrate, a silicon wafer chip, a flow through chip, a microbead, a nanoparticle, a polytetrafluoroethylene substrate, a polystyrene substrate, a gallium arsenide substrate, a gold substrate, and a silver substrate.

### Claim 27

A kit comprising:
a) one or more aptamers, wherein each of the one or more aptamers has specific binding affinity for one or more targets;
b) one or more solid supports;
c) one or more partitioning reagents;
d) one or more reagents for the release of an aptamer from an aptamer-target affinity complex;
e) one or more buffer solutions comprising an organic solvent; and
f) one or more buffer solutions comprising a chaotropic salt.

### Claim 28 (depends on 27)

The kit of claim 27, wherein said organic solvent is glycerol.

### Claim 29 (depends on 27)

The kit of claim 27, wherein said chaotropic salt is sodium perchlorate.

### Claim 30 (depends on 27)

The kit of claim 27 further comprising a reagent to cleave a cleavable moiety of the one or more aptamers.

### Claim 31

A method comprising:
contacting an aptamer having a specific affinity for a target molecule with a test sample, wherein an aptamer-target affinity complex is formed if the target molecule is present in the test sample, and wherein the aptamer is immobilized on a first solid support and washed with one or more solutions that dissociate aggregated aptamers;
removing one or more components not immobilized on the first solid support;
attaching a second tag to the target molecule in the aptamer-target affinity complex, wherein the second tag has an affinity to a second capture element;
releasing the aptamer-target affinity complex from said first solid support;
exposing the released aptamer-target affinity complex to a second solid support comprising a second capture element and allowing the second tag to interact with said second capture element to immobilize the aptamer-target affinity complex on the second solid support;
removing one or more components not immobilized on the second solid support; and
eluting the aptamer from the second solid support with one or more solutions comprising a chaotropic salt;
wherein, the target molecule is a protein.

### Claim 32 (depends on 1)

The method of claim 1, further comprising a kinetic challenge selected from a method comprising diluting the mixture containing the aptamer affinity complex; adding a competitor to the mixture containing the aptamer affinity; or a combination thereof.