Methods and/or Use of Oligonucleotide Conjugates for Suppressing Background Due to Cross-Hybridization

• US 20130344508A1
• Filed: 05/20/2013
• Published: 12/26/2013
• Est. Priority Date: 04/18/2007
• Status: Active Grant

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.), University of Chicago
• Original Assignee: Solulink, Incorporated (Maravai Life Sciences Holdings LLC), University of Chicago
• Inventors: Schwartz, David A., Williams, Jimmy, Zhao, Xinfang, Kron, Stephen J., Flor, Amy Catherine
• Granted Patent: US 9,222,936 B2
• Time in Patent Office: Days
• US Class Current: 435/7.21
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/547 with antigen or antibody attached to the carrier via a bridging agent

Abstract

The present disclosure is directed to methods and/or uses of oligonucleotide conjugates for assays and detections and related systems and/or kits for suppressing background due to cross-hybridization. Certain methods are directed to a method for detecting one or more biological targets of a sample in a detection assay, comprising: providing a molecular probe, comprising a binding moiety and an oligonucleotide sequence, to a sample comprising one or more biological targets; binding the one or more biological targets with the binding moiety; providing a detectable component to the sample, wherein the detectable component comprises a signal generating moiety conjugated to an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe; hydridizing the oligonucleotide sequence of the target-bound molecular probe to the detectable component; and detecting a signal generated from the hydridized detectable component. Various other embodiments, applications etc. are disclosed herein.

Claims

1. A method for suppressing cross-hybridization in assaying one or more targets of a sample, comprising:

i) hybridizing;

1) a molecular probe, comprising a binding moiety conjugated to an oligonucleotide sequence; and

2) a detectable component, comprising a signal generating moiety conjugated to an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe;

ii) providing to the hybridized molecular probe-detectable component a blocking component, comprising;

1) an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe;

or

2) an oligonucleotide sequence complementary to the oligonucleotide sequence of the detectable component;

iii) combining the blocked hybridized molecular probe-detectable component with one or more blocked hybridized molecular probe-detectable components similarly prepared by steps i) through ii);

iv) combining the mixture of the two or more blocked hybridized molecular probe-detectable components and the sample comprising the one or more targets;

v) binding the one or more targets in the sample with the binding moieties of the molecular probes of said two or more blocked hybridized molecular probe-detectable components;

vi) detecting a signal generated from the one or more targets bound to the molecular probes of said two or more blocked hybridized molecular probe-detectable components;

wherein the method is characterized by one or more of the following;

a) the conjugation between the oligonucleotide sequence and the binding moiety and conjugation between the complementary oligonucleotide sequence and the signal generating moiety, comprises one or more covalent bond linkages, comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates are at least 90% efficient; and

b) the binding moiety comprises a binding affinity of less than 10<sup>−

4</sup> M for the target.

2. The method of claim 1, wherein the assay comprises a multiplex assay.

3. The method of claim 1, wherein the assay detects, measures, or quantifies the level of binding and/or amount of the target present in the sample with one or more of the following:

flow cytometry, immunomagnetic cellular depletion, immunomagnetic cell capture, array, bead array, multiplex bead array, microarray, antibody array, cellular array, chemiluminescence, infrared, microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through said sample, increased light transmittance through said sample, a blotting method, a Western blot, a Southern blot, a Southwestern blot, labeling inside an electrophoresis system, labeling on a surface, labeling on an array, PCR amplification, elongation followed by PCR amplification, immunoprecipitation, co-immunoprecipitation, chromatin immunoprecipitation, pretargeting imaging, therapeutic agent, or combinations thereof.

4. The method of claim 2, wherein the assay detects, measures, or quantifies the level of binding and/or amount of the target present in the sample with flow cytometry.

5. The method of claim 1, wherein the sample is characterized as at least one or more of the following:

i) a complex sample; and

ii) a homogeneous or a heterogeneous mixture; and

wherein said sample comprises at least one or more of the following;

a) a range of analytes having a wide range of binding specificities;

b) a cell, a membrane, a biological molecule, a metabolite, or a disease biomarker; and

c) a biological fluid or a fluidized biological tissue.

6. The method of claim 1, wherein the blocking component comprises an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe.

7. The method of claim 6, wherein the blocking component is provided in an amount in the range of 1 to 200 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

8. The method of claim 6, wherein the blocking component is provided in an amount of 10 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

9. The method of claim 6, wherein the blocking component is provided in an amount of 25 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

10. The method of claim 6, wherein the blocking component is provided in an amount of 50 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

11. The method of claim 1, wherein the blocking component comprises an oligonucleotide sequence complementary to the oligonucleotide sequence of the detectable component.

12. The method of claim 11, wherein the blocking component is provided in an amount in the range of 1 to 200 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

13. The method of claim 11, wherein the blocking component is provided in an amount of 10 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

14. The method of claim 11, wherein the blocking component is provided in an amount of 25 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

15. The method of claim 11, wherein the blocking component is provided in an amount of 50 times greater than the concentration of the oligonucleotide sequence of the molecular probe.

16. The method of claim 1, wherein the method suppresses cross-hybridization in the range of 10% to 100% relative to a method exclusive of using a blocking component.

17. The method of claim 1, wherein the detectable component comprises a scaffold conjugated to the complementary oligonucleotide sequence, and wherein said scaffold has one or more signal generating moieties;

wherein the scaffold comprises a dendrimer, a polysaccharide, a dextran, a protein, a peptide, a second oligonucleotide sequence, a portion of the oligonucleotide sequence that is not complementary to the oligonucleotide sequence of the molecular probe, a polymer, a hydrophilic polymer, a bead, a nanoparticle, or combinations or derivatives thereof.

18. The method of claim 1, wherein the binding moiety comprises an antibody, a monoclonal antibody, a polyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a small molecule, an aptamer, a chelator, or combinations or derivatives thereof.

19. The method of claim 1, wherein the biological target comprises an antigen, a pathogen, a protein, a peptide, an epitope, a carbohydrate-containing molecule, a small molecule, or combinations or derivatives thereof.

20. The method of claim 1, wherein the signal generating moiety comprises:

a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, a fluorescent protein, a biofluorescent protein, a luminescent species, a chemiluminescent compound, a electrochemiluminescent label, a bioluminescent label, a phosphorescent species, a fluorophore labeled DNA dendrimer, Quantum Dot, a Raman particle, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactive isotope, a nanoparticle, a light scattering nanoparticle or microsphere, a diffracting particle, a polymer, a polymer particle, a bead, a solid surface, a metal particle, a stable isotope, a heavy metal chelate, a magnetic particle, an RFID tag, a microbarcode particle, an enzyme, an enzyme substrate, a molecule specifically recognized by another substance carrying a label or reacts with a substance carrying a label, an antibody, an antibody fragment, an antigen, a nucleic acid, a nucleic acid analog, oligonucleotide, oligonucleotide analog, complementary oligonucleotide, complementary oligonucleotide analog, a ligand, a protein, a peptide ligand, a protein substrate, a receptor;

a substrate, a secondary reporter, a hapten, or a combination or derivative thereof.