Methods and/or Use of Oligonucleotide Conjugates for Assays and Microscopy/Imaging Detections

Abstract

The present disclosure is directed to methods and/or uses of oligonucleotide conjugates for assays and microscopy/imaging detections and related systems and/or kits. 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.

1 . A method for assaying a target of a sample, comprising:
 i) providing to the sample:
 1) a molecular probe, comprising a binding moiety conjugated to a first oligonucleotide sequence; and 
 2) a detectable component, comprising a signal generating moiety conjugated to a second oligonucleotide sequence that is complementary to the first oligonucleotide sequence of the molecular probe; 
   ii) binding the target in the sample with the binding moiety of the molecular probe;   iii) hybridizing the oligonucleotide sequence of the molecular probe with the second oligonucleotide sequence of the detectable component; and   iv) detecting a signal generated from the hybridized detectable component using one or more of the following systems: microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC), or combinations thereof;   
       wherein the method is characterized by one or more of the following:
 a) the conjugation between the first oligonucleotide sequence and the binding moiety and conjugation between the second 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 strong binding affinity for the target. 
 
     
     
         2 . The method of  claim 1 , wherein the mode of addition comprises:
 i) the molecular probe and the detectable component are combined together and hybridized prior to contacting the sample;   ii) the molecular probe is combined with the sample prior to the addition of the detectable component; or   iii) the detectable component is combined with the sample prior to the addition of the molecular probe.   
     
     
         3 . The method of  claim 1 , wherein the method comprises:
 i) the molecular probe binding the target prior to hybridizing with the detectable component; or   ii) the molecular probe hybridizing with the detectable component prior to binding the target.   
     
     
         4 . A method for assaying a target of a sample, comprising:
 i) providing to the sample:
 1) a molecular probe, comprising a binding moiety conjugated to a first oligonucleotide sequence; 
 2) a detectable component, comprising a signal generating moiety conjugated to a second oligonucleotide sequence; and 
 3) a universal adapter, comprising an oligonucleotide sequence having a first sequence segment complementary to the first oligonucleotide sequence of the molecular probe and a second sequence segment complementary to the second oligonucleotide sequence of the detectable component; 
   ii) binding the target in the sample with the binding moiety of the molecular probe;   iii) hybridizing the first oligonucleotide sequence of the molecular probe to the first oligonucleotide sequence segment of the universal adapter;   iv) hybridizing the second oligonucleotide sequence of the detectable component to the second oligonucleotide sequence segment of the universal adapter; and   v) detecting a signal generated from the hybridized detectable component using one or more of the following systems: microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunohistochemistry (IHC), immunocytochemistry (ICC), or combinations thereof;   
       wherein the method is characterized by one or more of the following:
 a) the conjugation between the first oligonucleotide sequence and the binding moiety and conjugation between the second 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 strong binding affinity for the target. 
 
     
     
         5 . The method of  claim 4 , wherein the mode of addition comprises:
 i) the molecular probe, the universal adapter, and the detectable component are combined together and hybridized prior to contacting the sample;   ii) the molecular probe and the universal adapter are combined together and hybridized prior to contacting the sample;   iii) the detectable component and the universal adapter are combined together and hybridized prior to contacting the sample;   iv) the molecular probe, alone or in combination with the detectable component, is combined with the sample prior to the addition of the universal adapter; or   v) the universal adapter is combined with the sample prior to the addition of the molecular probe and/or the detectable component.   
     
     
         6 . The method of  claim 4 , wherein the method comprises:
 i) the molecular probe hybridizing with the universal adapter prior to said molecular probe binding the target;   ii) the molecular probe hybridizing with the universal adapter after said molecular probe binds the target;   iii) the detectable component hybridizing with the universal adapter prior to the molecular probe binding the target;   iv) the detectable component hybridizing with the universal adapter after the molecular probe binds the target;   v) the universal adapter hybridizing with the molecular probe and hybridizing with the detectable component prior to said molecular probe binding the target; or   vi) the universal adapter hybridizing with the molecular probe and hybridizing with the detectable component after said molecular probe binds the target.   
     
     
         7 . The method of  claim 1 , wherein:
 i) the assay comprises a singleplex or multiplex assay; and   ii) the assay further 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, immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through said sample, increased light transmittance through said sample, in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, 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. 
   
     
     
         8 . The method of  claim 1 , wherein the method carries out the detecting, measuring, or quantifying via microscopy, imaging, high content screening (HCS), or combinations thereof. 
     
     
         9 . The method of  claim 1 , wherein the method further comprises preparing and isolating the molecular probe, comprising:
 i) providing the binding moiety;   ii) conjugating the binding moiety with at least one first oligonucleotide sequence at greater than 90% efficiency to form binding moiety-oligonucleotide conjugates; and   iii) isolating the binding moiety-oligonucleotide conjugates from the conjugation mixture by binding, retaining, and/or retarding a substantial portion of:
 a) the conjugates, removing a substantial portion of the unconjugated first oligonucleotide sequence in a wash step followed by release of the bound, retained, and/or retarded conjugates; or 
 b) the unconjugated first oligonucleotide sequences, followed by collecting a substantial portion of the non-bound, non-retained, and/or non-retarded conjugates in a wash step. 
   
     
     
         10 . The method of  claim 9 , wherein the isolation step utilizes an immobilized binder, chromatography, affinity chromatography, size exclusion chromatography, HPLC, reverse-phase chromatography, electrophoresis, capillary electrophoresis, polyacrylamide gel electrophoresis, agarose gel electrophoresis, free flow electrophoresis, differential centrifugation, thin layer chromatography, immunoprecipitation, hybridization, solvent extraction, dialysis, filtration, diafiltration, tangential flow filtration, ion exchange chromatography, hydrophobic interaction chromatography, or combinations thereof. 
     
     
         11 . The method of  claim 1 , wherein the method further comprises preparing and isolating the detectable component, comprising:
 i) providing a plurality of the signal generating moiety;   ii) conjugating the second oligonucleotide sequence with at least one of the plurality of the signal generating moiety at greater than 90% efficiency to form signal generating moiety-second oligonucleotide conjugates; and   iii) isolating the signal generating moiety-second oligonucleotide conjugates from the conjugation mixture by binding, retaining, and/or retarded a substantial portion of:
 a) the conjugates, removing a substantial portion of the unconjugated second oligonucleotide sequences in a wash step followed by release of the bound, retained, and/or retarded conjugates; or 
 b) the unconjugated second oligonucleotide sequences, followed by collecting a substantial portion of the non-bound, non-retained, and/or non-retarded conjugates in a wash step. 
   
     
     
         12 . The method of  claim 1 , wherein the detectable component comprises a scaffold conjugated to the second oligonucleotide sequence, and wherein said scaffold comprises one or more signal generating moieties. 
     
     
         13 . The method of  claim 1 , wherein the scaffold comprises a dendrimer, a polysaccharide, a dextran, a protein, a peptide, a further oligonucleotide sequence, a portion of the second oligonucleotide sequence that is not complementary to the first oligonucleotide sequence of the molecular probe, a polymer, a hydrophilic polymer, a bead, a nanoparticle, or combinations or derivatives thereof. 
     
     
         14 . 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. 
     
     
         15 . The method of  claim 1 , wherein the target is a biological target. 
     
     
         16 . The method of  claim 15 , 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. 
     
     
         17 . The method of  claim 1 , wherein the signal generating moiety or the one or more signal generating moieties of the detectable component or the hybridized detectable component, comprises one or more of the following:
 a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, 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 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 Raman particle, 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 combinations or derivatives thereof.   
     
     
         18 . The method of  claim 1 , wherein the method comprises an automated system or robotic system. 
     
     
         19 . The method of  claim 1 , wherein the method further comprises removing the hybridized detectable component or plurality of detectable components from the bound target or plurality of targets, respectively, wherein said removal is by a washing or stripping process. 
     
     
         20 . The method of  claim 19 , wherein the method further comprises re-probing with a second detectable component or second plurality of detectable components, respectively, wherein said second detectable component comprises at least one second signal generating moieties conjugated to a second oligonucleotide sequence or a complementary second oligonucleotide sequence, or said second plurality of detectable components are prepared by independently pairing, via conjugation, a second plurality of signal generating moieties and a second plurality of second oligonucleotide sequences or a second plurality of complementary second oligonucleotide sequences.