Methods, Systems, and/or Use of Oligonucleotide Conjugates to Develop Panels for Use in Assays and Detections

Abstract

The present disclosure is directed to methods systems, and/or uses of oligonucleotide conjugates to develop panels for use in assays and 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 panel development system, comprising:
 i) a first panel comprising a plurality of molecular probes, said plurality of molecular probes prepared by independently pairing, via conjugation, a plurality of binding moieties and a plurality of oligonucleotide sequences;   ii) a second panel comprising a plurality of detectable components, said plurality of detectable components prepared by independently pairing, via conjugation, a plurality of signal generating moieties and a plurality of complementary oligonucleotide sequences;   iii) contacting a sample comprising a plurality of targets with the first panel and the second panel, wherein:
 a) at least a first target having one or more distinct bindings sites is bound by one or more molecular probes from the first panel; and 
 b) one or more detectable components from the second panel independently hybridize with the one or more molecular probes bound to the at least first target; 
   iv) detecting the presence of the at least first hybridized-target in said sample;   v) determining a characteristic panel of molecular probes and detectable components from said first and second panels that identifies the at least first target among the plurality of targets in said sample.   
     
     
         2 . The panel development system of  claim 1 , wherein the system comprises at least one or more of the following:
 i) the first panel comprises at least 2-10 different molecular probes;   ii) the second panel comprises at least 2-10 different detectable components;   iii) the first panel comprises between 2-40 different oligonucleotide sequences among the plurality of first oligonucleotide sequences; and   iv) the second panel comprises between 2-40 different second oligonucleotide sequences among the plurality of second oligonucleotide sequences.   
     
     
         3 . The panel development system of  claim 1 , wherein the panel development system further comprises a re-probe panel comprising a second plurality of detectable components, said second plurality of detectable components prepared by independently pairing, via conjugation, a second plurality of signal generating moieties and a second plurality of complementary oligonucleotide sequences. 
     
     
         4 . The panel development system of  claim 1 , wherein the panel development system further comprises removing the hybridized plurality of detectable components from the bound plurality of targets, wherein said removal is by a washing or stripping process. 
     
     
         5 . The panel development system of  claim 1 , wherein the panel development system further comprises a re-probing step comprising the contacting of the washed plurality of bound targets with said re-probe panel, followed by an additional detection step and determination of a characteristic panel of molecular probes and detectable components from said first, second, and re-probe panels that identifies the at least first target among the plurality of targets in said sample. 
     
     
         6 . A panel development system, comprising:
 i) a first panel comprising a plurality of molecular probes, said plurality of molecular probes prepared by independently pairing, via conjugation, a plurality of binding moieties and a plurality of first oligonucleotide sequences;   ii) a second panel comprising a plurality of detectable components, said plurality of detectable components prepared by independently pairing, via conjugation, a plurality of signal generating moieties and a plurality of second oligonucleotide sequences;   iii) a third panel comprising a plurality of universal adapters, said plurality of universal adapters comprising oligonucleotide sequences having independently paired a plurality of oligonucleotide sequence segments complementary to the plurality of first oligonucleotide sequences and a plurality of oligonucleotide sequence segments complementary to the plurality of second oligonucleotide sequences;   iii) contacting a sample comprising a plurality of targets with the first panel and the second panel, wherein:
 a) at least a first target having one or more distinct bindings sites is bound by one or more molecular probes from the first panel; and 
 b) one or more detectable components from the second panel independently hybridize with the one or more molecular probes bound to the at least first target; 
   iv) detecting the presence of the at least first hybridized-target in said sample;   v) determining a characteristic panel of molecular probes and detectable components from said first and second panels that identifies the at least first target among the plurality of targets in said sample.   
     
     
         7 . The panel development system of  claim 6 , wherein the panel development system is characterized by one or more of the following:
 i) the plurality of first oligonucleotide sequences are identical oligonucleotide sequences;   ii) the plurality of first oligonucleotide sequence segments are identical oligonucleotide sequence segments;   iii) the plurality of second oligonucleotide sequences comprises different oligonucleotide sequences; and   iv) the plurality of second oligonucleotide sequence segments comprises oligonucleotide sequence segments complementary to the plurality of different second oligonucleotide sequences.   
     
     
         8 . The panel development system of  claim 6 , wherein the panel development system is characterized by one or more of the following:
 i) the plurality of first oligonucleotide sequences comprises different oligonucleotide sequences;   ii) the plurality of first oligonucleotide sequence segments comprises oligonucleotide sequence segments complementary to the plurality of different first oligonucleotide sequences;   iii) the plurality of second oligonucleotide sequences are identical oligonucleotide sequences; and   iv) the plurality of second oligonucleotide sequence segments are identical oligonucleotide sequence segments.   
     
     
         9 . The panel development system of  claim 1 , wherein the plurality of binding moieties in said first panel comprises between 2-40 different binding moieties among the plurality of binding moieties. 
     
     
         10 . The panel development system of  claim 1 , wherein the plurality of signal generating moieties in said second panel comprises between 2-40 different signal generating moieties among the plurality of signal generating moieties. 
     
     
         11 . The panel development system of  claim 1 , wherein:
 i) the panel development system develops a singleplex or multiplex assay; and   ii) the panel development system and the assay developed by the panel development system 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 systems:
 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. 
   
     
     
         12 . The panel development system of  claim 1 , wherein the sample comprises a plurality of cells or cell types, comprising tissue cells, cells cultured in vitro, recombinant cells, infected cells, cells from laboratory animals, cells from mammal patients, cells from human patients, mesenchemal stem cells, stem cells, immuno-competent cells, adipose cells, fibroblasts, natural-killer cells (NK-cells), stem cells, monocytes, lymphocytes, lymph node cells, T-cells, B-cells, exudate cells, effusion cells, cancer cells, blood cells, comprising red blood cells, leukocytes, or white blood cells, organ cells, skin cells, liver cells, splenocytes, kidney cells, intestinal cells, lung cells, heart cells, or neuronal cells. 
     
     
         13 . The panel development system of  claim 1 , wherein the sample is characterized by or comprises at least one or more of the following:
 i) a plurality of similar targets or similar target types;   ii) a plurality of different targets or different target types;   iii) a plurality of cells having 2-50 different cell types;   iv) a plurality of different targets or different target types, wherein among a plurality of different targets in the sample at least a first target having one or more distinct bindings sites is bound by one or more molecular probes from the first panel;   v) at least 2-50 different targets or target types;   vi) the targets or target types comprises cells and/or cell types; and   vii) the targets or target types comprises cells and/or cell types comprising one or more distinct binding sites, distinct markers, or distinct biomarkers.   
     
     
         14 . The panel development system of  claim 1 , wherein the system further comprises at least one or more of the following:
 a) measuring the amount of the at least first target in said sample;   b) quantifying the level of the at least first target in said sample;   c) identifying the type of the at least first target in said sample; and   d) analyzing and/or identifying a sub-population of targets within the sample by assigning an immunophenotype resulting from signal pattern generated.   
     
     
         15 . The panel development system of  claim 1 , wherein one or more of the plurality of signal generating moieties 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, a bead, 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 thereof.   
     
     
         16 . The panel development system of  claim 1 , wherein the second panel comprises a plurality of detectable components, said plurality of detectable components prepared by independently pairing, via conjugation, a plurality of scaffolds and a plurality of complementary oligonucleotide sequences, wherein said plurality of scaffolds comprises a plurality of signal generating moieties. 
     
     
         17 . The panel development system of  claim 1 , wherein the scaffold comprises a dendrimer, a polysaccharide molecule, 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 bead, a polymer, a hydrophilic polymer, a bead, a nanoparticle, or combinations or derivatives thereof. 
     
     
         18 . The panel development system 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 panel development system of  claim 1 , wherein the panel development system comprises an automated system or robotic system. 
     
     
         20 . A multiplexed flow cytometry assay method, comprising:
 i) contacting a sample comprising a plurality of cells with a first series of molecular probes and a second series of detectable components, wherein the plurality of cells comprises at least 5 different cell types;   ii) binding protein markers on the plurality of cells with said first series;   iii) hybridizing the first series or the protein marker-bound first series with the second series; and   iv) optionally, identifying the cell types in the sample by assigning of immunophenotypes resulting from the hybridization of said protein marker-bound first series and said second series.