Protein-oligonucleotide compositions, kits, and methods thereof

Abstract

Examples are directed to composition, kits, and methods thereof. An example composition comprises a protein and an oligonucleotide linked to the protein via a tetrazine and a trans-cyclooctene (TCO) in a predetermined relationship.

Description

PROTEIN-OLIGONUCLEOTIDE COMPOSITIONS, KITS, AND METHODS THEREOFCross-Reference to Related Application

[0001] This application claims the benefit of US Provisional Application No. 63 / 551 ,168, filed February 8, 2024, which is incorporated herein by reference in its entirety.Reference to Sequence Listing

[0002] The contents of the electronic sequence listing(X1671111111_SequenceListing.xml; Size: 8,385 bytes; and Date of Creation: February 9, 2025) is herein incorporated by reference in its entirety.Background

[0003] Antibody-oligonucleotide compositions may be used for a variety of purposes, such as for an analytical test. Non-limiting example uses include different diagnostics and therapeutics. For example, an antibody may be configured to bind to a target analyte and an oligonucleotide may be used to provide a signal for detection. Conjugating antibody to oligonucleotides may rely on functional groups, such as lysines and cysteines, which exist in multiple places and may cause many degrees of conjugation and mixed species.Brief Description of the Drawings

[0004] FIG. 1 illustrates an example composition comprising a protein and an oligonucleotide.

[0005] FIGs. 2A-2D illustrate different example variations of compositions comprising a protein and an oligonucleotide.

[0006] FIGs. 3A-3C illustrate example uses of a protein-oligonucleotide conjugate and / or an imaging strand.

[0007] FIG. 4 illustrates an example of a plurality of protein-oligonucleotide conjugates.

[0008] FIGs. 5A-5C illustrate example kits which may be used to form a protein- oligonucleotide conjugate.

[0009] FIGs. 6A-6B illustrate further example kits which may include a protein- oligonucleotide conjugate.

[0010] FIGs. 7A-7B illustrate example protein-oligonucleotide conjugates.

[0011] FIG. 8 illustrates example mobility modulation techniques for protein- oligonucleotide conjugates.

[0012] FIG. 9 illustrates an example method of using a protein-oligonucleotide composition.

[0013] FIGs. 10A-10D illustrate example devices including non-transitory computer-readable medium storing executable instructions.

[0014] FIGs. 11A-11 B illustrate an example user interface including displays which may be provided by the device of FIG. 10A.

[0015] FIGs. 12A-12B illustrate an example user interface including displays which may be provided by the device of FIG. 10B.

[0016] FIGs. 13A-13C illustrate an example user interface including displays which may be provided by the device of FIG. 10C.

[0017] FIGs. 14A-14C illustrate an example user interface including displays which may be provided by the device of FIG. 10D.

[0018] FIGs. 15A-15C illustrate example protein-oligonucleotide conjugates or portions thereof.

[0019] FIGs. 16A-16G illustrate further variations of compositions comprising a protein and an oligonucleotide

[0020] FIGs. 17A-17F illustrate example an example multimer comprising a primary strand and complementary strand, with FIGs. 17B-17F showing different example primary strands.

[0021] FIG. 18 illustrates an example self-assembled protein-oligonucleotide (SAPO) multimer.

[0022] FIGs. 19A-21 are images from an example imaging reagent-application implementation.

[0023] FIG. 22 is an image of a gel from a mobility modulation reagentapplication implementation.

[0024] FIGs. 23A-23B and FIG. 24 are images of gels from tetrazine (tet) incorporation implementations.

[0025] FIG. 25 is an image of a gel from a tet incorporation implementations with protein A and one tet or two tets.

[0026] FIGs. 26A-26C are images of gels showing protein-oligonucleotide conjugates (PrOCs) with one protein and one oligonucleotide.

[0027] FIGs. 27A-27C are images of gels showing mobility of the PrOCs with one protein and one oligonucleotide.

[0028] FIGs. 28A-28B are images of gels showing mobility of the PrOCs as previously described by FIGs. 26A and 27A.

[0029] FIG. 29 is an image of a gel showing mobility of a multimer comprising a PrOC with one protein and three oligonucleotides linked via a multimer linker.

[0030] FIGs. 30A-30B are images of gels showing mobility of a multimer comprising a self-assembled protein-oligonucleotide (SAPO) with three PrOCs and a primary strand (PS).

[0031] FIGs. 31 A-31 B are images of gels showing mobility of a multimer comprising a SAPO with two PrOCs, one imaging strand (IS), and a PS.

[0032] FIGs. 32A-32B are images of gels showing mobility of a multifluor formed using a multimer linker.

[0033] FIGs. 33A-33B are images of gels showing mobility of multifluors comprising a SAPO.

[0034] FIGs. 34A-34B show Tables 8 and 9 that describe the reagents and procedures for forming each of the PrOC constructs or multifluor constructs.Detailed Description

[0035] In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific examples in which the disclosure may be practiced. It is to be understood that other examples may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present disclosure is defined by theappended claims. It is to be understood that features of the various examples described herein may be combined, in part or whole, with each other, unless specifically noted otherwise.

[0036] Antibody-oligonucleotide conjugates (AOCs) are an emerging class of chimeric biomolecules offering transformative applications in imaging, detection, and therapeutics. As noted above, antibody to oligonucleotide conjugation may rely on functional groups that exist in multiple places, which may result in many degrees of conjugation and mixed species. As such, AOCs often suffer from varying degree of conjugation (DOC), where the number of oligonucleotides attached to each protein is inconsistent and difficult to control. The lack of control may be attributed to the conjugation approaches and the types of chemistries used. Additionally, many current chemistry techniques for conjugation take a considerable amount of time and have substantial costs for forming the conjugate and require purification which may cause loss of functionality of the proteins. Further, target analytes that are closely resembled may be difficult to separate. For example, current approaches for creating these conjugates often rely on modifying existing antibodies through functional groups, including amines, carboxylic acids, sulfhydryls, and carbohydrates. Multiple and variable amine, carboxylic acids and sulfhydryls groups may be present on a given protein, such as an antibody and from antibody to antibody. Conjugation through the two carbohydrates groups is considered site-specific and more controllable. However, not all proteins or protein fragments contain these carbohydrate groups, such as certain single-domain antibodies (sdAb), antibody fragments (Fab), single-chain variable fragments (scFv), fluorescent proteins, and enzymes.A significant need exists for protein-oligonucleotide conjugates, which are not limited to antibodies but include antibodies, with precisely controlled DOC to enable applications that otherwise would be difficult or impossible to achieve using existing conjugates. The present invention addresses this need and provides additional advantages.

[0037] Examples in accordance with the present disclosure include protein- oligonucleotide conjugates which are formed from a protein and anoligonucleotide that are linked through a tetrazine (tet) and a trans-cyclooctene (TCO) in a predetermined relationship, sometimes referred to as “cardinality”. For example, the predetermined relationship may include a ratio of protein to tet that is predefined (e.g., 1 :1 , 1 :n, p: 1 , or p:n) and results in a predefined ratio of protein to oligonucleotide (e.g., p:o). That is, in some examples, the predetermined relationship may include a predefined ratio of protein to oligonucleotide. The ratio of protein to tet may be the same or different from the ratio of protein to oligonucleotide, in different examples. Because of the predetermined relationship, the resulting species may be identical and / or are not mixed. Furthermore, the use of tet may increase the speed of the reaction to form the protein-oligonucleotide conjugate, improve quantitation-stoichiometry, and improve purity as excess protein and / or oligonucleotide may not be needed to complete the reaction and which may result in no purification step being used in some examples. In some examples, the protein may include a single domain antibody (sdAb) (e.g., a nanobody) such that there is a smaller linkage error and high spatial resolution as compared to a larger protein. A protein that includes an sdAb may provide site specific linkage and cardinality. In some examples, the tet may be introduced as part of and / or incorporated into the protein.

[0038] Examples of the present disclosure are directed to compositions comprising a protein and an oligonucleotide. The compositions may be configured to bind to a target analyte, such as via the protein or the oligonucleotide. The target analyte may be in solution or in other sample forms including, but are not limited to, cell wall membrane, tissue sample, interstitial fluid, etc. Example target analytes may include biomarkers. As described above, the protein and oligonucleotide may form a conjugate. In some examples, the protein is bound to the tet and the oligonucleotide is bound to the TCO. In other examples, the protein is bound to the TCO and the oligonucleotide is bound to the tet. In any such examples, the TCO and tet may react such that the TCO is bound to the tet and the oligonucleotide is linked to the protein to form a protein- oligonucleotide conjugate. Such compositions may be used to form complexes with a target analyte and may be easily detected, without the use of complexsystems. For example, the compositions and formed complexes may be detected using standard microscopy technology.

[0039] The oligonucleotide may be linked to the protein via a tet and a TCO in a predetermined relationship. In some examples, the predetermined relationship of the tet to protein may include or be associated with a ratio of the protein to tet. For example, the ratio of protein to tet (and thus, the resulting ratio of protein to oligonucleotide) may include 1 :1. In other examples, the ratio of protein to tet includes 1 :n or p:1 , where n and / or p is greater than 1 . In some examples, p is less than n. In some examples, the ratio of protein to tet includes p:n, wherein p and n are the same number and are greater than 1 (e.g., 2:2, 3:3, 5:5) or are different numbers (e.g., 1 :2, 2:1 , 1 :3, 3:1 ). In some examples, p is less than n. By having a predetermined relationship of the protein to tet, the resulting conjugate species from reacting a plurality of proteins and oligonucleotides may be uniform, e.g., all containing the same number of oligonucleotide(s) linked to protein(s). As noted above, in some examples, the predetermined relationship may include or result in a ratio of protein to oligonucleotide (p:o).

[0040] As used herein, a protein refers to or includes a molecule comprising chains of amino acids, and which may fold into a three-dimensional structure. Proteins are not limited to full proteins and may include functional protein fragments (e.g., sdAb, single-chain variable fragment (scFv) antibodies, antibody fragments (Fab), mini-proteins (e.g., generated in silico), etc.). A nanobody refers to or includes an antibody fragment consisting of a singlevariable antibody domain, which may also be referred as a single domain antibody (sdAb). An oligonucleotide refers to or includes a sequence of nucleotides (e.g., a polynucleotide), which is short, such as containing less than 500 nucleotides. The oligonucleotide may be single stranded and / or double stranded, and may comprise deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) nucleotides. As used throughout, in some examples, a tet-modified protein may include or be referred to as a tetrazine-modified functional protein fragment.

[0041] As used herein, a target analyte refers to or includes a complex, molecule, or portion thereof (e.g., epitope) that binds to a ligand, such as theprotein conjugated to the oligonucleotide. In some examples, the target analyte may include multiple epitopes, and plurality of different compositions and / or portions of a composition may be configured to bind to different ones of the multiple epitopes. A ligand refers to or includes a molecule that binds to another molecule. A signal construct refers to or includes a molecule, multiple molecules and / or components which include or are bound to a signal component and are configured to be linked to a complex formed by the protein-oligonucleotide composition binding to the target analyte and used to detect the binding thereof and / or for other analytic outputs. A signal component refers to or includes any element (e.g., a molecule, component or object, dye, fluorophore, etc.) that provides or is used to derive a detectable signal or otherwise aid in detection. For example, the signal construct may include a fluorophore, a multifluor, dye, an oligonucleotide or multiple, a particle, or a magnetic component, as well as any combination thereof. The multifluor refers to or includes a collection of fluorophores that may absorb and / or emit the same or different colors. A label may include a molecule that is linked to or otherwise forms part of the complex including the target analyte and is used to aid in detection. A detectable label may itself output a detectable signal. In some examples, the label may react with another molecule to provide the detectable signal. The detectable signal may include and / or be derived from a wavelength, a charge, a mass, among other signals. A particle refers to or includes a material formed in a three- dimensional shape, such as a sphere, an ellipsoid, oblate spheroid, and prolate spheroid shapes, among other shapes such as irregular shapes. The particle may provide the detectable signal or be functionalized to exhibit the detectable signal. In some examples, the particle forms at least part of or includes the signal construct, such as including a dye or fluorophore. In some examples, the particle (e.g., the three-dimensional shape) and / or the detectable signal may not be visible to the human eye. For example, the detectable signal may be visible using a detection system, such as with fluorophores, and / or may be activated to be visible. A conjugate refers to or includes a compound that is formed of at least two compounds (or species) joined (e.g., covalently or other interaction) together. Conjugation refers to or includes formation of covalent bonds or otherinteractions which directly or indirectly (e.g., through another linker(s)) link at least two compounds.

[0042] Example are not limited to labels. For example, the signal construct may have an associated charge and / or mass, and may cause different target analytes, or at least one complex formed therefrom, to have distinguishable charge and / or mass, such that different target analytes and / or complexes may be separated by their charge and / or mass and then detected, such as by staining the target analytes and / or complexes for gel or electrically separating. In some examples, the signal construct is the oligonucleotide, the second oligonucleotide, or the second oligonucleotide with other signal components (e.g., a detectable label, a third oligonucleotide, a particle, among other components).

[0043] FIG. 1 illustrates an example composition comprising a protein and an oligonucleotide. In some examples, the composition 100 may include a protein- oligonucleotide conjugate.

[0044] As shown by FIG. 1 , the composition 100 comprises a protein 102. In some examples, the protein 102 is configured to bind to a target analyte 109. For example, the protein 102 may comprise a ligand that binds to the target analyte 109. A ligand may include a molecule that binds to a target analyte or other target. A target analyte includes the molecule or portion thereof (e.g., an epitope) that is being detected and / or measured, such as biomarkers of diseases, protein of a virus or other pathogens and / or other compounds.

[0045] In some examples, the protein 102 may include an antibody. In some examples, the protein may include an sdAb. Example antibodies includes full- size antibodies (e.g., two heavy chains and two light chains held together by disulfide bonds), enzymes, single domain antibodies, such as a single variable domain heavy chain (VHH), single-chain variable fragment (scFv) antibodies, or antibody fragments (Fab), among others. Example sdAb include a VHH. In other examples, the protein may include a non-antibody protein and / or a non-sdAb protein, such as an enzyme (e.g., Cas9, alkaline phosphatase, carbonic anhydrase, and horseradish peroxidase). In some examples, the non-antibody protein may include assay signal proteins, such as a green fluorescent protein,a red fluorescent protein, a yellow fluorescent protein, luciferase, or other peptides such as glucagon like peptide-1 (GLP-1).

[0046] The protein 102 may be a variety of different types of proteins and protein fragments, and are not limited to immunoglobulin G (IgG) or IgM. For example, a functional fragment of a protein may be used. As another example, sdAb may be used. Use of proteins that are different from IgG may allow for faster manufacturing. In some examples, the protein 102 may be made in cultures other than mammalian cells, such as E.coli cells, which may be faster (e.g., 3 times faster than mammalian cells) and less expensive (e.g., 1000 times less expensive than mammalian cells). Proteins may be prepared using different methods, (e.g., in cellulo, cell free, etc.)

[0047] The composition 100 further comprises an oligonucleotide 108. In some examples, the oligonucleotide 108 is configured to bind to the target analyte 109, such that the oligonucleotide 108 may be referred to as an aptamer. In some examples, the oligonucleotide 108 is single stranded. In some examples, the oligonucleotide 108 is double stranded. In some examples, the oligonucleotide 108 is a hybrid, which includes portion(s) that are double stranded and portion(s) that are single stranded. Further, the oligonucleotide 108 may include DNA and / or RNA.

[0048] In some examples, the oligonucleotide 108 (and optional second oligonucleotide) includes a length of between about 1 nucleotide and about 500 nucleotides or about 2 nucleotides and about 500 nucleotides. In some examples, the oligonucleotide 108 includes a length of between about 1 nucleotide and about 100 nucleotides. Examples are not so limited and may include other lengths, such as between about 2 nucleotides and about 500 nucleotides, about 1 nucleotide and about 400 nucleotides, about 1 nucleotide and about 300 nucleotides, about 2 nucleotide and about 250 nucleotides, about 1 nucleotide and about 200 nucleotides, about 1 nucleotide and about 150 nucleotides, about 4 nucleotides and about 500 nucleotides, about 4 nucleotides and about 350 nucleotides, about 4 nucleotides and about 300 nucleotides, about 4 nucleotides and about 250 nucleotides, about 4 nucleotides and about 200 nucleotides about 4 nucleotides and about 150nucleotides, about 4 nucleotides and about 100 nucleotides, about 4 nucleotides and about 80 nucleotides, about 4 nucleotides and about 70 nucleotides, about 4 nucleotides and about 65 nucleotides, about 4 nucleotides and about 50 nucleotides, about 4 nucleotides and about 25 nucleotides, about 4 nucleotides and about 10 nucleotides, about 15 nucleotides and about 500 nucleotides, about 75 nucleotides and about 500 nucleotides, about 100 nucleotides and about 500 nucleotides, about 150 nucleotides and about 500 nucleotides, about 20 nucleotides and about 500 nucleotides, about 50 nucleotides and about 350 nucleotides, about 50 nucleotides and about 250 or about 75 nucleotides and about 150 nucleotides, among other ranges.

[0049] In some examples, the oligonucleotide 108 (and optional second oligonucleotide) includes a length of between about 30 nucleotides and about 65 nucleotides. In some examples, the oligonucleotide 108 (and optional second oligonucleotide) includes a length of between about 30 nucleotides and about 70 nucleotides. In some examples, the oligonucleotide 108 (and optional second oligonucleotide) includes a length of between about 20 nucleotides and about 80 nucleotides. In some examples, the oligonucleotide 108 (and optional second oligonucleotide) includes a length of between about 5 nucleotides and about 100 nucleotides. In some examples, the oligonucleotide 108 (and optional second oligonucleotide) includes a length of between about 5 nucleotides and about 500 nucleotides.

[0050] The composition 100, via the protein 102 or the oligonucleotide 108, may be designed to bind to a variety of different target analytes. In some examples, the target analyte may be a biomarker of a disease (e.g., cancer), biomarker of a pathogen, such as a virus, bacteria, or other microorganism that may cause disease in humans or in other organisms, such as other animals and / or plants, among other organisms. In some examples, the target analyte 109 may include a small molecule, such as carbon dioxide (CO2). In some such examples, the protein 102 may include an enzyme that may be used as a catalyst, where the function of the composition 100 is to increase loading of the enzyme (e.g., carbonic anhydrase to convert CO2). The protein 102 (e.g., an enzyme) maycatalyze a reaction, such as binding to the target analyte 109 to cause the reaction to happen faster.

[0051] The oligonucleotide 108 is linked to the protein 102 via a tet 104 and a TCO 106 and in a predetermined relationship. The predetermined relationship may include or be associated with a ratio of the protein 102 to tet 104. For example, the ratio of protein 102 to tet 104 (and thus, the resulting ratio of protein 102 to oligonucleotide 108 due to the tet:TCO reaction) may include 1 :1. In other examples, the ratio of protein 102 to tet 104 includes 1 :n or p:1 , where n and / or p is greater than 1 . For example, at least one of n and p may be greater than 1 , such as n being greater than 1 , p being greater than 1 , or both n and p being greater than 1 . In some examples, the ratio of protein 102 to tet 104 includes p:n, wherein p and n are the same number or are different numbers. In some examples, p and / or n may each be between 1 and 50. In some examples, p and / or n may each be between 2 and 50, 5 and 50, 10 and 50, 25 and 50, 30 and 50, 40 and 50, 1 and 40, 1 and 30, 1 and 25, 1 and 10, 1 and 5, 2 and 40, 2 and 30, 2 and 25, 2 and 10, 2 and 5, 5 and 50, 5 and 25, 5 and 10, 10 and 50, 10 and 25, or 15 and 25, among other ranges. By having a predetermined relationship of the protein 102 to tet 104, the resulting species from reacting a plurality of proteins and oligonucleotides may be uniform, e.g., all containing the same number of oligonucleotide(s) linked to a protein and / or the same number of protein(s) linked to an oligonucleotide.

[0052] As noted above, in some examples, the predetermined relationship may include or be associated with a ratio of the protein 102 to oligonucleotide 108. In some examples, the ratio of protein 102 to oligonucleotide 108 includes 1 :1. In some examples, the ratio of the protein 102 to oligonucleotide 108 includes p: 1 , 1 :o, or p:o. In some examples, o and / or p is greater than 1 . For example, at least one of p and o may be greater than 1 , such as p being greater than 1 , o being greater than 1 , or both p and o being greater than 1 . In some examples, p and / or o may each be between 1 and 50, such as between 2 and 50, 5 and 50, 10 and 50, 25 and 50, 30 and 50, 40 and 50, 1 and 40, 1 and 30, 1 and 25, 1 and 10, 1 and 5, 2 and 40, 2 and 30, 2 and 25, 2 and 10, 2 and 5, 5 and 50, 5 and 25, 5 and 10, 10 and 50, 10 and 25, or 15 and 25, among other ranges.

[0053] As shown by FIG. 1 , in some examples, the protein 102 is bound to the tet 104 and the oligonucleotide 108 is bound to the TCO 106, with the TCO 106 being bound to the tet 104. For example, the TCO 106 may be covalently attached to an end of the oligonucleotide 108. The TCO 106 may react with the tet 104, thereby forming the bond. In some examples, the TCO 106 may be covalently bound to the tet 104 after reacting. Examples are not so limited and may include the protein 102 being bound to the TCO 106 and the oligonucleotide 108 bound to the tet 104. In any such example, the oligonucleotide 108 is linked to the protein 102 via the tet 104 and the TCO 106 reaction and in the predetermined relationship, regardless of whether the protein 102 is bound to the tet 104 or bound to the TCO 106 as the tet:TCO reaction may be 1 :1 or near 1 :1 , as further described herein.

[0054] As further described herein, examples are not limited to the oligonucleotide 108 being linked to the protein 102. Various examples are directed to kits which include the oligonucleotide 108 and the protein 102 which are configured to bind (e.g., to be linked) via the tet-TCO reaction to form the composition 100 (e.g., a conjugate), but which may be separate components. Such kits may include a plurality of components (e.g., oligonucleotide(s) and proteins(s) which may be selectively combined to generate different compositions, and may be referred to as an “experimental tool kit” or a “pretested kit”, as further described herein. Further, as used herein, linked refers to or includes direct and indirect linkages or other interactions, such as being indirectly linked through another linker or component. Further, linked includes bonds, such as covalent bonds, and other interactions, such as but not limited to annealing. In some example, components which are linked may be referred to as being “operatively coupled to”.

[0055] In some examples, the protein 102 may incorporate the tet 104 (or TCO 106), thereby forming a modified protein, e.g., tet-modified protein or TCO- modified protein. In some examples, the protein 102 incorporates the tet 104 such that the protein 102 may be said to contain a tet moiety, as further described herein. For example, the tet 104 may be incorporated into the protein 102 as a non-canonical amino acid via genetic code expansion, cell freesynthesis, or other types of protein systems. While not illustrated by FIG. 1 , in some examples, the protein 102 may incorporate at least two tets, as further described herein.

[0056] In some examples, the tet 104 (or TOO 106) is introduced into the protein 102. In such examples, the protein 102 is designed to have predetermined number of lysine and / or cysteine moieties for introducing the tet 104, such that the oligonucleotide 108 is linked to the protein 102 via a tet 104 and a TCO 106 and in a predetermined relationship. For example, the tet 104 may be introduced to the polypeptide chain of the protein 102 via coupling reactions with predetermined thiol group(s) or amine group(s) on the protein 102, such as through an addition of a predetermined number of cysteine or lysine residue(s) in the protein 102.

[0057] In some examples, the TCO 106 may react with the tet 104 via the inverse electron demand Diels-Alder (IEDDA) reaction, and may optionally form a protein-oligonucleotide conjugate. This reaction may be referred to as a type of “click chemistry” and is compatible with buffers suitable for biological workflows. The reaction is a second order reaction with a high reaction rate. In some examples, and in addition and surprisingly, the conversion rate and yield of the composition 100 (e.g., a protein-oligonucleotide conjugate) is high in buffers required for imaging and at room temperature and in significantly shorter time than anticipated. Additionally, the incubation time is relatively short, such as being about one hour or less to form the composition 100 at or near room temperature. Due to such features of the reaction, surprisingly, the composition 100 may be formed without use of a purification step to remove unreacted reagents. In some examples, the reaction is at a conversion rate and yield such that there is inconsequential amounts of the reagents remaining (e.g., unreacted reagents) after the reaction is complete, which may be due to the tet:TCO reaction being at or near 1 :1. Unreacted reagents may interfere with the results of later downstream processes of an application to be performed. For example, unconjugated protein (e.g., protein 102 not bound to the oligonucleotide 108) may bind to a target analyte and compete with the composition 100, thereby reducing the resulting signal and biasing the output. Similarly, excessoligonucleotide (e.g., oligonucleotide 108 not bound to the protein 102) that is not washed away may interfere with the signal, such as by binding to a complementary second oligonucleotide and competing with the composition 100.

[0058] In some examples, the oligonucleotide 108 may be quantitatively loaded on the protein 102 via the tet:TCO reaction. In some examples, when X mole of protein 102 bound to tet 104 (or protein 102 bound to TCO 106) are mixed with X mole of oligonucleotide 108 bound to TCO 106 (or oligonucleotide 108 bound to tet 104), the resulting volume of composition 100 formed may include X mole of composition 100 (e.g., protein 102 linked to oligonucleotide108). In some examples, the tet:TCO reaction may include a 1 :1 reaction. In some examples, the yield may be controlled by the adding a lesser amount of oligonucleotide 108 bound to TCO 106 (or oligonucleotide 108 bound to tet 104) or lesser amount of the protein 102 bound to tet 104 (or protein 102 bound to TCO 106) respectively (e.g., the oligonucleotide 108 bound to TCO 106 is in excess as compared to the protein 102 bound to tet 104 or vice versa). For example, mixing 0.5 mole of protein 102 bound to tet 104 with 1.0 mole of oligonucleotide 108 bound to TCO 106 may result in 0.5 mole of the composition 100 illustrated by FIG. 1.

[0059] The design of the protein 102 to contain a predetermined number of tet 104 or TCO 106 via incorporation of nonconical amino acid or genetic code expansion and / or via design of the predetermined number of cysteine or lysine residue(s), in various examples, allows for forming a volume of the composition 100 which have a controlled degree of conjugation, and thus a resulting volume of the composition 100 formed may include uniform species. Degree of conjugation may refer to the number of oligonucleotide(s) bound to the number of protein(s), such as 1 protein: o oligonucleotides or p proteins: 1 oligonucleotide, wherein o and / or p may be between 1 and 50. Although examples are not so limited, and in some examples, o and / or p may be greater than 50, such as with multimers. Uniformity of specifies may refer to the number of oligonucleotides bound to a protein or a number of proteins bound to an oligonucleotide being the same between each composition of the volume. Thecontrolled degree of conjugation and resulting uniformity of species may be due to the predetermined relationship, e.g., the ratio of the protein 102 to tet 104. For example, as the ratio of protein to tet is 1 : 1 , 1 :n, p:1 , or p:n, the reaction site (e.g., tet moieties) is set and results in the degree of conjugation (e.g., protein to oligonucleotide) of 1 :1 , 1 :o, p (e.g., p:1 ), or p:o. In some examples, the composition 100 may include multiple proteins per oligonucleotide or multiple oligonucleotides per protein to enhance the signal and / or enhance binding to the target analyte. Such compositions, which may be referred to as “multimers”, may be formed by using multiple reactive moieties, such as multiple tets, a linked multimer or other type of multimers, strained alkenes (e.g., TCO), and / or design of multiple cysteine or lysine residue(s) in the protein, as further described herein. In some multimers, the ratio of protein to tet may be the same or may be different from the ratio of protein to oligonucleotide.

[0060] In other design techniques that use click chemistry, the degree of conjugation is difficult and time consuming and challenging to control. For example, with strain-promoted azide and strainedalkyne click reaction (SPAAC), the azide and alkyne function groups on the protein and oligonucleotide facilitate conjugation. This second order reaction has a reaction rate that is several orders of magnitude slower than an inverse electron-demand Diels- Alder (lEDDA) reaction, and may have lower yield than the IEDDA reaction despite the longer incubation time. In some examples, the long incubation times may result in loss of function, because competing reactions cause the protein to aggregate, misfold or denature.

[0061] Additionally, the click functional group may need to be added to the protein prior to the SPAAC reaction to make the protein-oligonucleotide conjugate. This may be done in several ways. One way is to use existing lysine residues on the protein and react them with NHS ester chemistry. A drawback to this approach is that there are a variable number of lysine residues on different proteins. This means that the degree of conjugation is difficult to control. As noted above, examples using TCO and tet have set reaction site(s) (e.g., 1 , 1 / n, p, or p:n) and leads to a degree of conjugation of 1 , 1 / o, p, or p:o. Additionally,conjugating to native lysine may disrupt the structure of the protein leading to changes in stability and binding affinity.

[0062] The functional groups may also or alternatively be attached to native cysteines in protein. Here, the native disulfide bonds in the protein need to be cleaved to make the thiol group available for conjugation with maleimide chemistry to add the SPAAC functional group. While there are fewer cysteine compared to lysine, this approach also may lead to uncontrolled degrees of conjugation, protein instability, and reduced binding affinity.

[0063] A third conjugation site that other approaches may use is the two glycosylation sites on the protein, such as an antibody. The carbohydrate chains are modified to allow for click chemistries. This is considered a site-specific conjugation method because there is only one glycosylation site on each heavy chain of a full length antibody. However, with antibody fragments, such as VHH, Fab, and scFv, do not have these sites. While there may be some control over the degree of conjugation, there are still three distinct species produced and / or no other degree of conjugation available. For example, the three species may include unconjugated (e.g., no oligonucleotide linked to a protein), two sites (e.g., 2 oligonucleotides linked 1 protein), and one site (1 oligonucleotide linked to 1 protein). In accordance with various examples of the present disclosure, further freedom of degrees of conjugation is provided and results in a single species.

[0064] Additionally, such prior methods may require at least two purification steps, the first after adding the SPAAC functional group to the protein, and the second after conjugating the oligonucleotide. Example methods of forming the composition 100 including a tet 104 introduced or incorporated into the protein 102 in accordance with the disclosure may not require purification and therefore exhibit less (or no) loss of protein and may retain a higher yield.

[0065] Another approach is to use a non-canonical amino acid with an azide side chain to incorporate the SPAAC functional group. While this technique may be specific, it suffers from drawbacks of the SPAAC reaction with regard to slower reaction rates compared to IEDDA, the need for a purification step, and low yields. Purification may result in loss of protein (e.g., lower yield);additionally, the long incubation times may result in loss of function, because competing reactions cause the protein to aggregate, misfold or denature. As noted above, example reactions in accordance with the present disclosure may exhibit faster reaction times, may not require purification, and result in higher yields with uniform species compared to prior approaches.

[0066] The composition illustrated by FIG. 1 may include variations. Example variations include, but are not limited to, oligonucleotides being double stranded and / or single stranded, the protein incorporating the tet, and / or use of a second oligonucleotide which optionally forms at least part of or is configured to link to a signal construct, among other variations. At least some of the variations are further illustrated by FIGs. 2A-8 and 16A-18.

[0067] In some examples, the composition 100 may include additional components, such as but not limited to additional protein(s), oligonucleotide(s), and / or linker(s). As an example, the composition 100 may additionally include a multimer linker that links the oligonucleotide 108 to the protein 102 via the tet 104 and the TCO 106. In some examples, the multimer linker may be linked to the protein 102 via tet 104 and the TCO 106 (e.g., the TCO is directly or indirectly linked to the multimer linker), with the oligonucleotide 108 being (directly or indirectly) linked to the multimer linker, such as illustrated further herein by FIG. 16F. In some examples, the multimer linker may be linked to the protein 102 indirectly via tet 104, the TCO 106, and the oligonucleotide 108, such as further illustrated herein by FIG. 16E.

[0068] In some examples, the multimer linker comprises a plurality of branches. In some examples, one of the plurality of branches is linked (e.g., directly or indirectly) to the oligonucleotide 108 and a second of the plurality of branches is linked (e.g., directly or indirectly) to the TCO 106 and linked to the protein 102 via the tet 104 and the TCO 106, the composition 100 further including additional proteins linked to tet and linked to the remaining plurality of branches of the multimer linker via the tets and TCO. In some examples, one of the plurality of branches is linked (e.g., directly or indirectly to the oligonucleotide 108 (which is conjugated to the protein 102 via the tet 104 and TCO 106), the composition 100 further including additional proteins linked to tet and additionaloligonucleotides linked to TCO and which are linked (e.g., annealed) to the remaining plurality of branches of the multimer linker.

[0069] In some examples, the composition 100 comprises a self-assembled protein-oligonucleotide (SAPO) multimer comprising the protein 102 and the oligonucleotide 108, and a second oligonucleotide. As further described herein, the second oligonucleotide may be a primary strand and the oligonucleotide 108 is complementary to a portion of the primary strand, and the composition 100 further includes a third oligonucleotide that is complementary to another portion of the primary strand, and a second protein linked to the third oligonucleotide via another tet and TCO.

[0070] FIGs. 2A-2D illustrate different example variations of compositions comprising a protein and an oligonucleotide. Various features and attributes of the compositions 200, 201 , 203 of FIGs. 2A-2C may include at least substantially the same features and attributes of the composition 100 of FIG. 1 , as shown by the common numbering and with the details of the common features and attributes not being repeated. In some examples, any of the compositions 200, 201 , 203 of FIGs. 2A-2C may include an example implementation of the composition 100 of FIG. 1.

[0071] As shown by FIG. 2A, in some examples, the composition 200 includes a protein that contains (e.g., incorporates or is introduced) the tet, referred to in FIG. 2A as the “tet-modified protein” 210. The tet-modified protein 210 is linked to the oligonucleotide 208 via the tet of the tet-modified protein 210 and TCO 206, as previously described.

[0072] In some examples, the tet-modified protein 210 may be formed by introducing the tet into the polypeptide chain of the protein via coupling reactions with thiol or amine groups on the protein, or by incorporating the tet as a non-canonical amino acid via generic code expansion, cell free synthesis, or other types of protein synthesis. In such examples, the tet may be a non-conical amino acid that is incorporated into the polypeptide chain of the protein via generic code expansion, cell free synthesis, or other types of protein synthesis. In other examples, as previously described, the tet may be introduced into the polypeptide chain of the protein by modifying the protein to include apredetermined number of lysine or cysteine moieties in the polypeptide and coupling reactions with the tet(s).

[0073] In some examples, the tet-modified protein 210 may be prepared by genetic encoding using a non-canonical amino acid bearing the tet (e.g., a tetrazine moiety). For instance, the tet-modified protein 210 may be genetically encoded to include a ligand. Using an orthogonal aminoacyl-tRNA synthetase and an orthogonal tRNA, the non-canonical amino acid (in this case, a tet), a tet-modified protein 210 (which may be a tet-modified functional protein fragment) may be prepared that includes both the tet and the protein containing or that is the ligand. In some examples, the non-canonical amino acid includes salt forms thereof. In various examples, the protein includes a fragment or portion of a protein. For instance, a fragment of protein A may comprise the ligand, and the fragment of protein A may be genetically encoded to include the tet to generate the tet-modified protein 210.

[0074] For more general information on proteins that incorporate a tet, and specific information on example tet structures, reference is made to US Patent Publication 2019 / 0077776, published on March 14, 2019, and entitled “Reagents and methods for bioorthogonal labeling of biomolecules in living cells”, which is herein incorporated by reference in its entirety for its teachings.

[0075] In some examples, the tet-modified protein 210 covalently binds to the TCO 206, which may maintain avidity of the protein, sometimes referred to as the “orthogonality”. In some examples, multimers of the tet-modified protein 210 may be prepared, such as with one or more tets at a pre-selected location on the protein to control the length and orientation of the tet-modified protein 210 when linked to the oligonucleotide 208.

[0076] In some examples, as shown by FIG. 2B, the oligonucleotide 208 is double stranded. In such examples, the protein 202 is linked to the oligonucleotide 208 via the tet 204 of and the TCO 206, as previously described. In some examples, a double stranded oligonucleotide may be used to reduce steps (e.g., no annealing of the CS during the reagent-application) and / or costs (e.g., oligonucleotide below a threshold length, such as 60 single or double stranded base pairs, may be below a threshold cost). In someexamples, adding the CS may be used to increase the mass and / or charge without increasing the length of the PS.

[0077] In other examples, as shown by FIG. 2C, the oligonucleotide 208 is single stranded. In some such examples, a second oligonucleotide 212, which is also single stranded and is complementary to the oligonucleotide 208, may be used to detect binding between the protein 202 and a target analyte 209. In some examples, the second oligonucleotide 212 may form at least part of or may be configured to link to a signal construct (which may include a signal component), as further illustrated herein.

[0078] In some examples, a single stranded oligonucleotide may be used without a complementary second oligonucleotide. For example, for a mobility modulation reagent-application, the single stranded oligonucleotide may have sufficient mass and / or charge to allow for separation when linked to the target analyte 209. In other examples, a double stranded oligonucleotide may be used to provide the mass and / or charge.

[0079] In some examples, as shown by FIG. 2D, the oligonucleotide 208 is configured to bind to the target analyte 209. In such examples, the oligonucleotide 208 may be referred to as an aptamer. An aptamer refers to or includes a molecule designed or selected to be a ligand, and may include an oligonucleotide. Such examples may allow for further design freedom for the protein202. In any of the examples, the tet 204 (or multiple) may be incorporated into the protein 202 or the tet (or multiple) 204 may be introduced into the protein 202 via reactions of designed number(s) of lysines and / or cysteines. In various examples, an aptamer may be single stranded and / or double stranded, and may fold into a shape (similar to tertiary folding of a protein) configured to bind to the target analyte.

[0080] FIGs. 3A-3C illustrate example uses of a protein-oligonucleotide conjugate and / or imaging strands (IS). Various features and attributes of the protein-oligonucleotide conjugate 300 of FIGs. 3A-3B may include at least substantially the same features and attributes of the composition 100 of FIG. 1 and / or any of the compositions 200, 201 , 203 of FIGs. 2A-2D, as shown by the common numbering and with the details of the common features and attributesnot being repeated. In some examples, the protein-oligonucleotide conjugate 300 of FIGs. 3A-3B may include an example implementation of the composition 100 of FIG. 1 and / or any of compositions 200, 201 , 203 of FIGs. 2A-2D.

[0081] As shown by FIGs. 3A-3B, in some examples, the protein-oligonucleotide conjugate 300 includes a protein 302, a tet 304, a TCO 306, and a single stranded oligonucleotide 308. In some such examples, the single stranded oligonucleotide 308 may be referred to as a docking strand (DS). A DS is an oligonucleotide that is configured to be coupled to the target analyte 309, such as through the protein 302. As shown by FIG. 3A, in some examples, the protein-oligonucleotide conjugate 300 is exposed to a sample. In response to the sample containing the target analyte 309, in some examples, the protein 302 binds to the target analyte 309.

[0082] After exposing the protein-oligonucleotide conjugate 300 to the sample, a second oligonucleotide 312 is added, as shown by FIG. 3A. The second oligonucleotide 312 is single stranded and complementary to the oligonucleotide 308. In some examples, the second oligonucleotide 312 may form part of a signal construct 315, as further described below. In other examples, a separate signal construct 315 may be added which is configured to link to the second oligonucleotide 312, such as via linkers. The signal construct 315 may further include a signal component 314. In some examples, the second oligonucleotide 312 includes the signal component 314. In some examples, as illustrated by FIGs. 3A-3B, the second oligonucleotide 312 is linked to a first linker 313 and the signal construct 315 includes a second linker 316 configured to bind with the first linker 313. For example, the signal construct 315 may include the signal component 314 linked to a second linker 316. The first linker 313 and the second linker 316 may react to link the second oligonucleotide 312 to the signal component 314. In some examples, the signal component 314 includes a detectable label, such as a visual or optical label that output a visual or optical signal (e.g., a fluorophore, a dye). As shown by FIG. 3B, the oligonucleotide 308 and the second oligonucleotide 312 may anneal to one another, such that the signal component 314 is linked to the target analyte 309 via the protein- oligonucleotide conjugate 300.

[0083] The signal component 314 may be subsequently detected, which indicates the presence of the target analyte 309 in the sample. In some examples, the signal component 314 may provide an optical or visual signal which may be detected with a human eye and / or using imaging device and / or circuitry. In such examples, the (single stranded) second oligonucleotide 312 may be referred to as an imaging strand (IS). An IS is an oligonucleotide that is complementary to a DS and includes a dye, a fluorophore, a multifluor, or other signal component 314 which may be detected using imaging circuitry or equipment.

[0084] The signal construct 315 may provide, directly or indirectly, an indication that the target analyte 309 and the conjugate 300 are linked, either via the protein 302 or the oligonucleotide 308. In various examples, the signal construct 315 may include a molecule, complex, particle or other object that provides the detectable signal, e.g., an optical signal, a visual signal, an electrical signal, a magnetic signal, and / or an electromagnetic signal, among others. In some examples, the signal is optical, such as being optically or visibly detectable by a human or a machine. In some examples, a particle may provide the optical signal, such as with gold nanoparticles. The detectable signal includes or refers to a property or signal which may be detected or derived, such as a visual color, optical signal (e.g., fluorescence), electrical or magnetic property, radioactive property, among other labels which may be detected. In some examples, the signal component 314 is a fluorophore that outputs a fluorescent signal at a particular wavelength. In some examples, the signal component 314 is the second oligonucleotide 312 that causes a detectable change in charge and / or mass when the second oligonucleotide 312 anneals to the oligonucleotide 308, and which may be used to derive an output signal. In some examples, the signal component 314 is a particle that causes a change in mass and / or charge when the second oligonucleotide 312 anneals to the oligonucleotide 308.

[0085] A linker refers to or includes a molecule that binds to another molecule (such as another linker), and may be bound to the oligonucleotides and / or signal components. Example linkers include TCO including a TCO derivative, e.g., sTCO, with functional groups (e.g., moieties), for example, a TCO with anamine moiety, a TCO with a carboxylic acid moiety, a norbornene anhydride, a norbornene with an amine moiety, a multimer linker, a tet and / or a norbornene with a carboxylic acid moiety, among other molecules. For example, one of the first and second linkers 313, 316 may include another tet and the other of the first and second linkers 313, 316 may include another TCO. However, examples are not so limited and may include other types of linkers and / or other techniques for binding the signal component 314 to the second oligonucleotide 312. Similarly, examples are not limited to signal components that provide an optical signal. For example, the second oligonucleotide 312 annealing to the oligonucleotide 308 may cause a change (e.g., modulation) in charge and / or mass, which can be detected. In such examples, the second oligonucleotide 312 is itself the signal construct 315.

[0086] Some examples may include use of the protein-oligonucleotide conjugate 300 as illustrated by FIGs. 3A-3B for imaging, such as to detect the target analyte in a sample. An example imaging reagent application includes cyclic immunofluorescence (CycIF). CycIF is a multiplexed immunofluorescence imaging technique used to semi-quantitatively assess for the presence of target analytes (e.g., proteins) of interest in biological samples, such as biological tissue. For any type of immunofluorescence application, including but not limited to CycIF, the number of target analytes that can be imaged may be limited by the number of fluorescent channels on a microscope or other type of imaging circuitry. Microscopes are typically limited to three to four channels, with some commercially available instruments being capable of imaging six to seven channels. To view additional target analytes, CycIF is used to iteratively cycle through different sets of the plurality of target analytes using the limited number of channels.

[0087] In some specific examples, CycIF may involve use of a thin slice of tissue, referred to as a “section”, which is kept intact to observe tissue structures, cell types, and spatial relationships. For cancer, this can allow for researchers, physicians, or other type of users to determine what type of cancer is present, how much cancer is present, where in the tissue and / or organ thecancer has invaded, and / or how many immune cells are present in the normal and cancer regions.

[0088] The tissue may be fixed and embedded in paraffin wax or frozen from fresh tissue. The method may require removal of the wax after the section is attached to a microscope slide. Then, the antigen retrieval steps reverse the effects of fixation that make protein epitopes unavailable for protein binding. Additional preparation steps may be performed before adding primary antibodies. In some examples, the use of the protein-oligonucleotide conjugate 300 may reduce the incubation time for each cycle of the CycIF. For example, the protein-oligonucleotide conjugate 300 may be used as the DS, as is sometimes herein referred to as a protein-DS for ease of reference. All protein- DS may be added to the sample and incubated at one time to reduce the total incubation time, and with the associated IS then selectively added in different cycles, as described further below. As the total set of protein-DS for all target analytes are added at once, there is one total incubation for all target biomarkers, which may be 12-24 hours total instead of 12-24 hours per each of the number cycles (e.g., 12-24 hours multiplied by the number of cycles) as with other types of conjugates.

[0089] As a specific example of CycIF, assume there are four channels available and forty total target analytes. With these parameters, the CycIF includes ten cycles, with a subset of four target analytes in each cycle. Each of the four target analytes is associated with a protein-DS, e.g., protein- oligonucleotide conjugate, and an IS, e.g., a complementary oligonucleotide linked to or forming part of a signal construct, associated with one of the four channels (e.g., target 1 : channel 1 , target 2: channel 2, target 3: channel 3, target 4; channel 4). The IS being associated with a channel may include a second oligonucleotide including or being linked to the signal constructor molecule associated therewith) that exhibits a signal at the wavelength of or overlapping with the respective channel. During each cycle or iteration, the sample (e.g., slice of tissue) is exposed to a different set of associated protein- oligonucleotide conjugates, e.g., protein-DS, and a set of associated IS for the respective four target analytes, such that all forty target analytes are analyzedonce all cycles are performed. As may be appreciated, an associated protein- oligonucleotide conjugate includes a protein-oligonucleotide conjugate having a protein configured to bind to the target analyte, and the associated IS includes a complementary oligonucleotide to the oligonucleotide of the conjugate. Responsive to exposure to the set of four protein-DS and associated four IS in a respective cycle, if the four target analytes are present in the sample, the protein-DS binds to the target analytes and the IS anneals to the DS of the respective protein-DS. After an incubation period (e.g., 1-24 hours), unbound protein-DS and IS are washed away in a washing step, and then the sample is imaged for the four channels. The data may be analyzed (then or after all cycles), and the signals of the signal constructs are removed or inactivated for the next cycle to be performed. By removing or inactivating the signal, the same four channels may be used for each of the cycles.

[0090] In various examples and using the above example of four channels and forty target analytes, rather than exposing the sample to the protein-DS in subsets across the ten cycles, the sample may be exposed to all forty protein- DS at one time and incubated to allow for binding and then optionally unbound protein-DS are washed away in a washing step. In each cycle, the sample is then exposed to the four IS associated with four protein-DS of the respective cycle, and then incubated for about a 0.25 hour to about a 2 hour period of time. After the (small) incubation period, unbound IS are washed away in another (or a first) washing step and then the sample is imaged for the four channels. For example, an image is taken of each of the four fluorescent channels to capture the signals. Each IS anneals to a single protein-DS of the forty added and has a fluorophore that emits at one of the four channels (and which is unique from others in the cycle). The same set of fluorophores may be used for each cycle. If the four target analytes are present in the sample, the protein-DS bind to the target analytes during the first incubation, and the associated IS anneal to the DS during the subsequent incubation of the respective cycle. The data may be analyzed (then or after all cycles), excess IS is washed away, and the signals of the signal constructs are removed or inactivated for the next cycle to be performed. While the above describes two washing steps, in various examples,one washing step may be used after adding the four IS, and which may wash away unbound protein-DS, unbound IS, and protein-DS which are bound to IS but not bound to the target analyte. In some examples, the volume of IS added may be equal to or greater than the volume of protein-DS, and / or at a volume sufficient that binding between protein-DS and IS, without target analyte binding, is not a concern.

[0091] The signal may be removed or otherwise inactivated using a variety of techniques. In some examples, the signal may be removed using techniques such as thermal denaturation, restriction enzymes, chemical bleaching, photobleaching, disulfide bond cleavage with TCEP, strand mediated displacement, and / or use of a photocleavable linker, among other techniques. For example, thermal denature may be used to denature the oligonucleotide from the second oligonucleotide, but may not be suitable for single-step staining. A restriction enzyme may be used to break the oligonucleotide(s). Chemical bleaching may include (but is not limited to) use of NaHC HO2 oxidation to bleach the signal, but over many cycles may be destructive to reagents and (tissue) sample. In some examples, ultra-violet light may be used to cleave a photo-cleavable linker between the signal component (e.g., fluorophore) and the oligonucleotide. The cleaved components may be washed away, and the next cycle may then begin.

[0092] FIG. 3C illustrates an example IS which may be used with any of the example protein-oligonucleotide compositions and conjugates as described herein. In some examples, the IS may include a plurality of detectable labels 314-1 , 314-2 such that the detectable signal output is amplified.

[0093] In some examples, as shown by FIG. 3C, the IS 301 may include the second oligonucleotide 312 linked to two detectable labels 314-1 , 314-2, one at the 5’ end and one at the 3’ end of the second oligonucleotide 312.

[0094] Although FIG. 3C illustrates two detectable labels 314-1 , 314-2 linked to the IS 301 (or other type of oligonucleotide, such as a CS), examples are not so limited, and more than two detectable labels may be linked to an oligonucleotide using a variety of techniques, such as but not limited to linkers as previously described.

[0095] Using the above-described protein-oligonucleotide conjugates and design of the imaging process, in various examples, between three to eight channels may be used to assay between 1 to 200 or more target analytes across many different cycles. However examples are not so limited, and may include greater than eight channels. In an example imaging method, all protein- oligonucleotide conjugates may be added to the sample at one time and incubated to cause binding between the protein-oligonucleotide conjugates and target analytes present in the sample. Respective second oligonucleotides with signal components(e.g., IS) are added in each cycle, the first and second oligonucleotide anneal, the sample is imaged, and then the signal is removed or otherwise inactivated, which is repeated for all cycles and until all IS are added. In some examples, the types of fluorophores or other detectable label used may be adjusted for the anticipated concentration of target analytes. For example, if a low concentration of a particular target analyte is anticipated, a fluorophore that is brighter than other fluorophores may be used on the IS to increase the signal. Brightness of a fluorophore or other optical signal may be associated with its wavelength and may be determined by the extinction coefficient times Quantum yield / 1000. In some examples, fluorophore brightness may be classified on a scale, such as using the fluorophore brightness index score of 1 to 5 (e.g., 1 is dim and 5 is brightest).

[0096] In some examples, the protein-oligonucleotide composition may include a multimer that includes multiple proteins and / or multiple oligonucleotides. The multiple proteins and / or multiple oligonucleotides may include the same proteins or different proteins from one another and / or the same oligonucleotides or different oligonucleotides from one another. In some examples, the multimer may include the p:n relationship of protein to tet, where p and n are different numbers or are the same number that is greater than 1 . A multimer, as used herein, refers to or includes a composition that includes multiple proteins and / or multiple oligonucleotides. In some examples, the multimer may include a linker formed by the tet to TCO reaction, where the linker is formed by at least one tet to TCO reaction. Such multimers may be referred to as a “linked multimer”. In some examples, the multimer may include a linker formed by at least oneoligonucleotide, which may include branches or arms for linking to multiple proteins. Such example multimers may be referred to as a “self-assembled protein-oligonucleotide (SAPO) multimer”. The ratio of protein to tet of p:n in the multimer may result in a composition having a ratio of protein to oligonucleotide of p:o, wherein the ratio of p:n and p:o may be the same or may be different from one another. In various examples, the multimer may be implemented to include at least some of substantially the same features and attributes of any of the multimers as described further below.

[0097] FIG. 4 illustrates an example of a plurality of protein-oligonucleotide conjugates. The protein-oligonucleotide conjugates 401 , 403 may form part of a kit 400 which includes many different conjugates targeted to different target analytes 409-1 , 409-2. Each protein-oligonucleotide conjugate 401 ,403 includes a protein 402-1 , 402-2, a tet 404-1 , 404-2, a TCO 406-1 , 406-2, and an oligonucleotide 408-1 , 408-2, which is optionally configured to anneal to a second oligonucleotide 412-1 , 412-2, as previously described. While two conjugates 401 , 403 and two target analytes 409-1 , 409-2 (as well as two detectable labels 416-1 , 416-2) are illustrated, examples are not so limited and may include greater than two for each. In some examples, such as for assessing post translation modification or interrelated target analytes, the two protein-oligonucleotide conjugates 401 , 403 may be used where there is an intent to capture the two target analytes 409-1 , 409-2 which are on the same target molecule and assess each relative to the other. Various features and attributes of each of the protein-oligonucleotide conjugates 401 , 403 of FIG. 4 may include at least substantially the same features and attributes of the composition 100 of FIG. 1 , any of the compositions 200, 201 , 203 of FIGs. 2A- 2D, and / or conjugate 300 of FIGs. 3A-3B and / or IS of FIGs. 3C-3E, as shown by the common numbering and with the details of the common features and attributes not being repeated. In some examples, any of the protein- oligonucleotide conjugate 401 , 403 may include an example implementation of the composition 100 of FIG. 1 , any of compositions 200, 201 , 203 of FIGs. 2A- 2D, conjugate 300 of FIGs. 3A-3B, and / or IS of FIGs. 3C-3E, or the compositions of FIGs. 16A-18, among others.

[0098] In some examples, a kit, method, and / or other types of systems and devices may involve use of a multiple different and unique proteins for select targets. For example, as illustrated by FIG. 4, each of the protein- oligonucleotide conjugate 401 , 403 may be configured to respectively bind to a different target analyte 409-1 , 409-2. Further, in some examples, different signal components 416-1 , 416-2 may be used. In some examples, the different signal components 416-1 , 416-2 may include different fluorophores which output the optical signals at different wavelengths. In the particular example, the signal components 416-1 , 416-2 are linked to the second oligonucleotides 412-1 , 412- 2 via a linker 417-1 , 417-2 to form signal constructs 415-1 , 415-2.

[0099] The linker 417-1 , 417-2 may be the same or different linkers in examples. Further, although one linker is illustrated, the linkers 417-1 , 417-2 may include first and second linkers (e.g., tet and TCO) such as illustrated by FIGs. 3A-3B. Examples are not limited to signal components which provide optical signals, and may include other types of signal components.

[0100] Such kits having protein-oligonucleotide conjugates 401 , 403 with predetermined degree of conjugation may be used to form assays with less background signal (e.g., noise) and that are more quantitative compared to conjugates with uncontrolled or mixed degrees of conjugation. For example, with mixed degrees of conjugation, the ratio of protein to oligonucleotide is different among species and each binding event with a second oligonucleotide may cause emission of a different amount of signal, depending on the degree of conjugation. With the predetermined degree of conjugation, the amount of signal per species may be the same in some examples.

[0101] FIGs. 5A-5C illustrate example kits which may be used to form a protein-oligonucleotide conjugate. Various features and attributes of any of the kits 530, 540, 550 of FIGs. 5A-5C may include at least substantially the same features and attributes of the composition 100 of FIG. 1 , compositions 200, 201 , 203 of FIGs. 2A-2D, and / or any of the conjugates and / or IS of FIGs. 3A-4 as shown by the common numbering and with the details of the common features and attributes not being repeated.

[0102] In some examples, as shown by FIG. 5A, a kit 530 may comprise a protein 502 linked (e.g., bound) to a tet 504, and an oligonucleotide 508 linked (e.g., bound) to a TCO 506, wherein the TCO 506 is configured to react with (and bind to) the tet 504 thereby linking the oligonucleotide 508 to the protein 502. As previously described, in some examples, the tet 504 may be in a predetermined relationship with the protein 502. In some examples, the predetermined relationship includes or is defined by a ratio of protein to tet (or oligonucleotide) being 1 :1. In some examples, the predetermined relationship includes or is defined by a ratio of protein to tet being 1 :n or p:1 , wherein n and / or p is greater than 1 . In some examples, the predetermined relationship includes or is defined by a ratio of protein to tet being p:n. In some examples, the predetermined relationship includes or is defined by a ratio of protein to oligonucleotide, such as the ratio being 1 :1 , p:1 , 1 :o, or p:o, as previously described. In some example, predetermined relationship includes or is defined by a ratio of protein to oligonucleotide being 1 :o or p:1 , wherein o and / or p is greater than 1. In some examples, the protein 502 may incorporate the tet 504. In some examples, the tet 504 may be introduced into the protein 502, as previously described. In some examples, the protein 502 is an sdAb, an antibody (e.g., full size or fragment), or an enzyme, among other examples. In some examples, the oligonucleotide 508 is single stranded and / or double stranded, and / or is DNA and / or RNA. Furthermore, the TCO 506 may bind to the tet 504 to form a protein-oligonucleotide conjugate, as previously described.

[0103] Examples are not so limited. In some examples, as illustrated by FIG. 5B, a kit 540 may comprise a protein 502 linked to a TCO 506, and an oligonucleotide 508 linked to a tet 504, wherein the TCO 506 is configured to react with (and bind to) the tet 504. The kit of 540 (as well as kit 530 of FIG. 5A and kit 550 of FIG. 5C) may include any of the above described examples and / or variations, including those described above for kit 530 of FIG. 5A.

[0104] In some examples, the kit may include additional components. For example, the kit 550 of FIG. 5C may include the same features and attributes of the kit 530 of FIG. 5A, but with the addition of a second oligonucleotide 512. The second oligonucleotide 512 is complementary to the oligonucleotide 508,with both the oligonucleotide 508 and the second oligonucleotide 512 being at least partially single stranded and complementary to one another.

[0105] In some examples, the kit 550 may include a signal construct 515. In some examples, the signal construct 515 includes a signal component 516 that provides a detectable signal. In some examples, the signal construct 515 is the second oligonucleotide 512 which provides a change in charge and / or mass once bound to the oligonucleotide 508 which may be used to derive an output signal and / or use to separate different complexes, as further described herein. In some examples, the second oligonucleotide 512 includes a first linker 513 and the kit 550 further includes a signal construct 515 including a signal component 516 bound to second linker 514 configured to bind with the first linker 513 of the second oligonucleotide 512. In other examples, the signal construct 515 provided in the kit 550 may include the signal component 516 linked to the second oligonucleotide 512 via the first and second linkers 513, 514.

[0106] In some examples, the signal component 516 is a fluorophore, the second oligonucleotide 512 (e.g., a physical property, such as charge or mass), a particle, or a combination thereof, and is used to derive a signal from: a fluorescent signal, a change in mass, a change in charge, or a combination thereof. In some examples, the signal component 516 outputs a visual or optical signal. For example, the signal component 516 may include a fluorophore or a dye which is visually or optically detected. In some examples, the signal component 516 may include a particle, such as a dye or other types of particles. In some examples, a signal construct 515 may include multiple signal components, such as multiple of the same fluorophore or different fluorophores, among other signal components and combinations thereof. In some examples, the signal component 516 may change the charge and / or mass of the full complex, which may be used to derive an output signal, as further described herein.

[0107] As may be appreciated, any of the described kits may include different oligonucleotides and proteins which are separate from one another, but configured to be linked. As such, the terms “configured to bind” or “configured tolink” refers to or includes components that are presently separated but can be selectively reacted to link to one another. Such kits may include experimental tool kits or pre-tested kits that include the different components which may be used for different reagent-applications, such as for research, diagnostics, imaging, or other purposes. In some examples, the pre-tested kit may include a panel of components which are pre-defined for the specific reagent-application, such as a set of protein-oligonucleotide conjugates that have been tested for specific reagent-application, with an understanding that the compounds do not generally cross react.

[0108] Example kits may include additional components than illustrated by the kits 530, 540, 550 of FIGs. 5A-5C, such as additional proteins, oligonucleotides, TCOs or other linkers.

[0109] In some examples, a kit may further include a multimer linker. For example, the multimer linker may be configured to bind to the oligonucleotide and the protein via the tet and the TCO reaction (e.g., directly or through additional linkers). In some examples, the multimer linker comprises a plurality of branches, wherein one of the plurality of branches is configured to bind to the oligonucleotide and a second of the plurality of branches is linked to TCO and configured to bind to the protein via the tet and the TCO reaction, the kit further including additional proteins linked to tet and configured to bind to the remaining plurality of branches of the multimer linker. In some examples, the multimer linker comprises a plurality of branches, one of the plurality of branches being configured to bind (e.g., directly or indirectly) to the oligonucleotide, the kit further including additional proteins linked to tet and additional oligonucleotides linked to TCO and configured to bind to the remaining plurality of branches of the multimer linker. That is, although FIGs. 5A and 5C show a TCO directly bound to the oligonucleotide 508, examples are not so limited and the TCO may be directly bound to a branch of the multimer linker and indirectly bound to the oligonucleotide 508 after conjugation.

[0110] Any of the example kits may comprise components for a SAPO multimer including the protein, the oligonucleotide, and a second oligonucleotide. In some such examples, the second oligonucleotide is aprimary strand and the oligonucleotide is complementary to a portion of the primary strand, and the kit further includes a third oligonucleotide that is complementary to another portion of the primary strand, and a second protein linked to the third oligonucleotide via another tetrazine and TCO.

[0111] FIGs. 6A-6B illustrate further example kits which may include a protein- oligonucleotide conjugate. Various features and attributes of any of the kits 660, 670 of FIGs. 6A-6B may include at least substantially the same features and attributes of the composition 100 of FIG. 1 , compositions 200, 201 , 203 of FIGs. 2A-2D, any of the conjugates and / or IS of FIGs. 3A-4, and / or kits 530, 540, 550 of FIGs. 5A-5C, as shown by the common numbering and with the details of the common features and attributes not being repeated. In some examples, the kits 660, 670 of FIGs. 6A-6B may include an example implementations of any of the kits 530, 540, 550 of FIGs. 5A-5C.

[0112] As shown by FIG. 6A, an example kit 660 includes a conjugate 600 including a protein 602 conjugated to an oligonucleotide 608 through a tet 604 and a TCO 606 such that the tet 604 is in a predetermined relationship with the protein 602. In some examples, the protein 602 is bound to the tet 604. In some examples, the protein 602 incorporates the tet 604 to form a tet-modified protein, and in other examples, the tet 604 is introduced to the protein 602, as previously described. In some such examples, the oligonucleotide 608 may be bound directly to the TCO 606, which reacts with the tet 604 to form a bond. Examples are not so limited and may include different variations as described above, including the protein 602 being directly bound to the TCO 606 and the oligonucleotide 608 being bound directly to the tet 604, among other variations.

[0113] As previously described, the predetermined relationship may include a ratio of protein to tet, such as a ratio of 1 :1 , 1 :n, p:1 , or p:n; and / or the predetermined relationship may include a ratio of protein to oligonucleotide, such as a ratio of 1 :1 , 1 :o, p:1 , or p:o. The predetermined relationship may cause the same ratio of protein 602 to oligonucleotide 608 across species of the conjugate 600.

[0114] The kit 660 may further include a second oligonucleotide 612 that is complementary to the oligonucleotide 608 and which forms at least part of or isconfigured to link to a signal construct 615 in some examples. The signal construct 615 may include a signal component 616 configured to provide or otherwise derive a detectable signal. In some examples, the first and second oligonucleotides 608, 612, or at least a portion thereof, may be single stranded, and are each either DNA and / or RNA. As previously described, the signal construct 615 may include a fluorophore, a multifluor, a dye, the second oligonucleotide 612, a particle, a magnetic component, or any combination thereof, including but not limited to multiple of the same signal component (e.g., multiple of the same fluorophore).

[0115] In some examples, as illustrated by FIG. 6A, the second oligonucleotide 612 includes or is linked to a first linker 613 and the signal construct 615 includes a second linker 614 configured to bind / react with the first linker 613, as previously described. The second linker 614 may be bound to the signal component 616.

[0116] In some examples, kits may include a plurality of protein-oligonucleotide conjugates. For example, and as illustrated by FIG. 6B, a kit 670 may include a set of conjugates 671 , a set of second oligonucleotides 673, and a set of signal constructs 675, wherein the set of conjugates 671 each comprise different proteins 602-1 , 602-2, 602-N or oligonucleotides 608-1 , 608-2, 608-N configured to bind to a different target analyte. Each conjugate of the set of conjugates 671 includes a tet 604-1 , 604-2, 604-N, a TCO 606-1 , 606-2, 606-N, and an oligonucleotide 608-1 , 608-2, 608-N. Each oligonucleotide 608-1 , 608-2, 608-N may be different and may be complementary to a different second oligonucleotide 612-1 , 612-2, 612-N of the set of second oligonucleotides 673. Furthermore, each signal construct 615-1 , 615-2, 615-M may include signal components 616-1 , 616-2, 616-M that are different from others of the set of signal constructs 675 (or at least portions thereof). In some examples, the signal constructs 615-1 , 615-2, 615-M may include first linkers 614-1 , 614-2, 614-M for binding to the second oligonucleotides 612-1 , 612-2, 612-N or to second linkers bound to the second oligonucleotides 612-1 , 612-2, 612-N. In some examples, the second oligonucleotides 612-1 , 612-2, 612-N may be pre-bonded to respective ones of the signal components 616-1 , 616-2, 616-M prior toperforming a reagent-application, such as a test, and based on a design for the reagent-application, and thus forming signal constructs including the second oligonucleotides 612-1 , 612-2, 612-N linked to different ones of the signal components 616-1 , 616-2, 616-M, as further described herein.

[0117] More particularly, FIG. 6B illustrates the use of DS and IS for an imaging reagent-application, such as for diagnostics, treatment, and / or research. In such examples, the number (N) of oligonucleotides 608-1 , 608-2, 608-N may be the same as the number (N) of second oligonucleotides 612-1 , 612-2, 612-N, such that there is a unique complementary IS for each DS. The number (M) of signal constructs 615-1 , 615-2, 615-M and associated signal components 616-1 , 616-2, 616-M may be the same as N or may be less than N. For example, six fluorophores may be used to label sixty second oligonucleotides 612-1 , 612-2, 612-N (wherein N is 60 and M is 6) across ten cycles of an application, as further described herein.

[0118] Examples are not limited to imaging reagent-applications and may be used for other types of reagent-applications, including but not limited to, polymerase chain reaction (PCR) (e.g., ImmunoPCR), proximity ligation or extension assays, electrochemical proximity assays, super resolution microscope (e.g., DNA-Paint), drug delivery (e.g., siRNA delivery and oligonucleotide antivirals), in vivo imaging and / or other in vivo reagentapplications, protein arrays, immunohisto-chemistry, flow cytometry, covalent antibody display, functional biomaterials (e.g., hydrogels, nanofibers, extracellular matrix mimetics), self-assembling materials (e.g., nanoparticles, supramolecular deoxyribonucleoprotein), DNA nanostructures for antigen presentation, vaccine adjuvant, nucleic acid stability and aptamer conjugation, among others. In some examples, such as illustrated by FIGs. 7A-8, the detectable signal may be derived from a change in charge and / or mass, which may be used to detect the target analyte and / or to separate complex(es) by the charge and / or mass (e.g., to perform electrophoretic separation). For example, the oligonucleotide, and optionally the second oligonucleotide, may be between 4-500 (or more) nucleotides long, with each nucleotide carrying a -1 chargeand / or including modified nucleotides which changes the mass of the formed complex without necessarily changing the charge, as further described below.

[0119] FIG. 7A-7B illustrate further example protein-oligonucleotide compositions. Various features and attributes of the protein-oligonucleotide compositions 700 of FIGs. 7A-7B may include at least substantially the same features and attributes of the composition 100 of FIG. 1 and / or any of the compositions 200, 201 , 203 of FIGs. 2A-2D, as shown by the common numbering and with the details of the common features and attributes not being repeated. In some examples, any of the compositions 700 may include an example implementation of the composition 100 of FIG. 1.

[0120] As shown by FIG. 7A, the composition 700 includes a protein 702 linked to an oligonucleotide 708 via the tet 704 and TCO 706. In some examples, the oligonucleotide 708 is single stranded and may bind to a second oligonucleotide 712, thereby changing the mass and / or charge of the full complex including the composition 700 when linked to the target analyte 709. In such examples, the second oligonucleotide 712 may be referred to as the signal construct 715 including the signal component. In some examples, the oligonucleotide 708 may be double stranded (and / or single stranded) and may provide the change in mass and / or charge of the complex formed therefrom and to provide separation from another target analyte or other complexes formed from other compositions in a kit. The complex may include the target analytes 709 linked to the composition 700 linked to the second oligonucleotide 712 (and optionally, the detectable label). For ease of reference, the oligonucleotide 708 is sometimes herein referred to as the “primary strand (PS)”, which is the oligonucleotide strand that may be linked to the protein 702 and / or, in some examples, is complementary to second oligonucleotide(s). The optional second oligonucleotide 712 is sometimes referred to as the “complementary strand (OS)”, which is the oligonucleotide strand configured to anneal to the PS.

[0121] In some examples, the second oligonucleotide 712 may include additional components, as shown by FIG. 7B. For example, a particle 718 (or other signal component which aids in signal detection through optical orstructural changes (e.g., which provides a change in mass and / or charge) may be linked (e.g., bound) to the second oligonucleotide 712 through a linker 714.

[0122] In any of the examples, the signal components (e.g., 708, 712, 718) may provide a change in mass and / or charge to the complex formed by the target analyte 709, composition 700, and, in some examples, second oligonucleotide 712 which is different from other changes in mass and / or charge from at least one another target analyte and / or from other complexes formed from a kit (e.g., a plurality of different compositions configured to bind to different target analytes and having different signal components). By providing different changes in mass and / or charge, the complexes may be distinguishable from one another and may allow for the target analytes (forming part of the complexes) to be separated via electrophoretic separation. For example, FIG. 8 provides example modulation techniques 880 which may be used to perform electrophoretic separation of one or more target analytes. For example, one target analyte may be separated out of an unknown sample. In some examples, at least two target analytes may be separated from one another.

[0123] As described in further detail below, the modulation techniques 880 may include: (i) varying a length of the PS and / or the CS to change a charge and / or mass of the complex; (ii) using a PS and / or CS with modulated nucleotides that are charge neutral, and of variable length, to change a mass of the complex; and / or (iii) using a PS, a CS, and a second CS which is complementary to the CS, and any of which may have variable lengths alone or in combination, to change the mass or charge of the complex. In some examples, the different modulation techniques 880 may be used in different combinations, such as to form different distinguishable complexes and to provide separation for a plurality of target analytes which may be close in mass and / or charge. In some examples, the third technique may be used iteratively during the reagent-application to provide further separation when needed, and as further described below.

[0124] In some examples, a method for performing electrophoretic separation may include adding a plurality of different protein-oligonucleotide conjugates which are designed to respectively provide different and distinguishablechanges in mass and / or charge from other protein-oligonucleotide conjugates of the plurality. The different and distinguishable changes in mass and / or charge may be used to separate complexes formed, and thus provide separation to a plurality of target analytes. In some examples, the plurality of different protein- PS may include single and / or double stranded oligonucleotides which provide the distinguishable changes in mass and / or charge. In some examples, the plurality of different protein-PS may include single stranded oligonucleotides and the method further includes adding a plurality of CS (e.g., second oligonucleotides). Examples are not so limited and may include methods which modify only one target analyte using a protein-oligonucleotide conjugate to provide mass or charge separation from a second target analyte and / or to separate the one target analyte from an unknown sample. Other variations may include the use of modified charge neutral nucleotides, third (or more) oligonucleotides, particles, and detectable labels, among others.

[0125] Accordingly, various examples include the use of single and / or double stranded oligonucleotides, e.g., PS, CS, and / or second CS, to add mass and / or charge to at least one target analyte in a convenient and modular way. The modularity is provided from the ability to make oligonucleotides of any length and / or controlled charge. The average base pair is 660 grams / mol or 660 Daltons (Da). If, for example, the objective is to increase a 15 kDa target protein in size by two times, then a 15 kDa double stranded oligonucleotide of 23 base pairs may be used. Nucleotides are larger than amino acids, so relatively small amounts of nucleotides may be added to change the protein mass upon configuration.

[0126] For charge modulation, each base pair add a -2 charge because of the phosphate backbone. Likewise, each single stranded base adds a -1 charge. For example, an oligonucleotide of 10 double stranded base pairs adds a -20 charge. To add less charge, single stranded oligonucleotides may be used.

[0127] In FIG. 8, the protein-oligonucleotide conjugates 803, 805 show examples of adjusting a length of the PS and / or CS to modulate the mass and / or change of the complex 884-3, 884-4 formed by the protein- oligonucleotide conjugates 803, 805, the target analytes 809-3, 809-4, and thesecond oligonucleotides 812-3, 812-4. For example, the second oligonucleotides 812-3, 812-4 may form the signal construct 815-3, 815-4 which causes the formed complex 884-3, 884-4 to change in mass or charge from the unmodified target analyte’s mass and / or charge, and to be distinguishable in mass or charge from another target analyte or from other complexes formed from other target analytes. For example, respective second oligonucleotides 812-3, 812-4 may anneal to the oligonucleotides 808-3, 808-4 to modulate the length of complexes 884-3, 884-4, resulting in complexes 884-3, 884-4 having different and distinguishable charges or masses from the other target analyte or from other complexes formed.

[0128] As may be appreciated, for either charge or mass, the length of a double stranded PS may be adjusted in some examples. In some examples, the length of the single stranded PS may be adjusted and / or the length of the single stranded CS may be adjusted. In some examples, as shown by the complex 884-4, the formed complex 884-4 may include an overhang of the CS (e.g., nucleotide bases of the second oligonucleotide 812-4 which extend past nucleotide bases of the oligonucleotide 808-4 and do not have complementary nucleotide bases from the oligonucleotide 808-4). In some examples, this overhang may be varied to provide different overhangs, e.g., the CS may bind at different locations to the PS, such that the single stranded CS may hang over at different lengths to provide the distinguishable changes in mass and / or charge. In some examples, additional signal components may be added to the CS, e.g., linking a detectable signal and / or a particle to further modulate the mass and / or charge.

[0129] For mass modulation, but without adding negative charge, modified nucleotide may be used. Examples of modified nucleotides include phosphodiamidate morpholino oligos (PMOs) or H-phosphonate. In some examples, the charged oxygen in the phosphate backbone may be replaced with an amide group or hydrogen yielding a neutral charge. The mass to charge ratio may be further changed by hybridizing strands of DNA with PMO or other modified oligonucleotides. In FIG. 8, the protein-oligonucleotide conjugate 801 shows an example of using a signal construct 815-2 including a secondoligonucleotide 812-2 with modified nucleotides to modulate the mass without adding negative charge. In the example, the complex 884-2 formed by the protein-oligonucleotide conjugate 801 , the target analyte 809-2, and the second oligonucleotide 812-2 includes an overhang of the second oligonucleotide 812-2 from the oligonucleotide 808-2. In some examples, this overhang may be varied, as described above. In some examples, there may be no overhang.

[0130] In some examples, an additional (e.g., third) oligonucleotide may be added to anneal to the overhang portion of the CS, which may be further used to provide the distinguishable change in mass and / or charge to a complex formed. In FIG. 8, the protein-oligonucleotide conjugate 800 shows an example of using a signal construct 815-1 including a second oligonucleotide 812-1 and a third oligonucleotide 820. The second oligonucleotide 812-1 is complementary and anneals to the oligonucleotide 808-1 linked to the protein 802-1 and the third oligonucleotide 820 is complementary and an anneals to the second oligonucleotide 812-1 (e.g., the overhang portion). In some examples, the length of the oligonucleotide 808-1 , the second oligonucleotide 812-1 , and / or the third oligonucleotide 820 may be varied to modify the mass and / or charge of the complex 884-1 formed by the protein-oligonucleotide conjugate 800 binding to the target analyte 809-1 and the second oligonucleotide 812-1 and third oligonucleotide 820 respectively annealing to the oligonucleotide 808-1 and the second oligonucleotide 812-1 . In some examples, the first, the second and / or third oligonucleotides 808-1 , 812-1 , 820 may be linked to a fluorophore or other detectable labels, such as illustrated by 882-1 , 882-2, and 882-3.

[0131] Changing mass and / or charge may be useful in electrophoresis assays, such as the electrophoretic mobility shift assay. For example, if two or more target analytes have the same or similar migration on a gel, the proteins appear as one band. Changing the mass and charge of at least one target analyte may result or cause the target analytes (e.g., at least two) to migrate at different lengths and appear as separate bands. In various examples, binding at least one target analyte (e.g., protein of interest) with a protein-PS provides the mass and / or charge difference to modulate the mobility within the mobility shift assay. Further, a desired mobility pattern may be achieved by finding the right mass tocharge protein-PS. For example, sdAb-oligonucleotide conjugates may be formed with varying ratios of mass and charge to separate target analytes (e.g., proteins of interest) with inherently similar mobilities. Furthermore, a combination of a protein-PS having a single stranded oligonucleotide and a CS may provide additional modulation. The CS may have a variable length, or be coupled to another mass and / or charge, such as a nanoparticle or to a label.

[0132] In any examples, after adding protein-oligonucleotide (e.g., protein-PS) and the optional second oligonucleotide (e.g., CS), the method may include separating complexes formed of the target analyte, protein-oligonucleotide, and optionally the second oligonucleotide. The separation may be based on mass and / or charge, and occurs by applying an electric field to move negatively charged complexes (or other molecules) through a matrix of agarose or other substance (e.g., gels, glass microfiber, cellulose membranes, resins, etc.). For example, a negative charge may be applied at one end of the matrix and a positive charge at the other, with complexes migrating to form distinct bands in the gel. Smaller complexes or complexes with greater charge may move faster than larger complexes or complexes with less charge on suitable matrices.

[0133] The formed complexes may be separated based on the distinguishable changes in charge and / or mass, and without washing away unbound oligonucleotides (e.g., protein-PS and / or CS). In some examples, different complexes formed of target analytes may provide changes in charge and / or mass which distinguish complexes formed from unbound protein-PS and / or CS. For example, the bands from protein-PS or protein-PS-CS may have distinguishable mass and / or charge from complexes formed including target analytes linked to protein PS and optionally CS. In some examples, the target analytes may be stained such that unbound protein-PS and / or CS may not provide a band.

[0134] FIG. 8 illustrates an example of a plurality of protein-oligonucleotide conjugates. Various features and attributes of any of the protein-oligonucleotide conjugates of FIGs. 8 may include at least substantially the same features and attributes of the composition 100 of FIG. 1 , any of the compositions 200, 201 , 203, 700 of FIGs. 2A-2D and / or FIGs. 7A-7B, any of the conjugates of FIGs. 3A-4, and / or any of the kits 530, 540, 550, 660, 670 of FIGs. 5A-6B, as shown by the common numbering and with the details of the common features and attributes not being repeated.

[0135] In various examples, a kit may include different types of protein- oligonucleotide conjugates 800, 801 , 803, 805 or variations thereof which may be used to form different kits. For example, one of the types (e.g., 800) may be used to form a kit by varying the number or length of the oligonucleotide(s) (e.g., the PS, CS, and / or third oligonucleotide), the detectable label, and / or other signal components for each target analyte. In some examples, a kit may include use of multiple modulation types and combinations thereof for different target analytes. For example, examples may include various combinations of the above modulation techniques, such as but not limited to, respective ones of the oligonucleotides being different lengths and / or the CS binding at different locations of the PS to provide different hangover regions, respective CS having additional components (e.g., particles), and respective CS being configured anneal to a third oligonucleotide, and with the set of differences being designed to provide the distinguishable changes in mass and / or charge for a plurality of complexes to be formed with a plurality of target analytes.

[0136] To illustrate the different modulation techniques 880, FIG. 8 shows example protein-oligonucleotide conjugates 800, 801 , 803, 805 which each include a protein 802-1 , 802-2, 802-3, 802-4 respectively configured to bind to a target analyte 809-1 , 809-2, 809-3, 809-4. In some examples, as previously described, the oligonucleotides may be configured to bind to target analytes. Each protein 802-1 , 802-2, 802-3, 802-4 is linked to an oligonucleotide 808-1 , 808-2, 808-3, 808-4 via a tet 804-1 , 804-2, 804-3, 804-4 and a TCO 806-1 , 806- 2, 806-3, 806-4. In some examples, each oligonucleotide 808-1 , 808-2, 808-3, 808-4 is complementary to a second oligonucleotide 812-1 , 812-2, 812-3, 812- 4. However, examples are not so limited, and in some examples, the 808-1 , 808-2, 808-3, 808-4 may be double stranded or single stranded, and may provide the change in mass or charge without the use of second oligonucleotide 812-1 , 812-2, 812-3, 812-4.

[0137] As noted above, in some examples, respective ones of the second oligonucleotides 812-1 , 812-2, 812-3, 812-4 may anneal to the oligonucleotides 808-1 , 808-2, 808-3, 808-4 to provide different length complexes 884-1 , 884-2, 884-3, 884-4, resulting in complexes 884-1 , 884-2, 884-3, 884-4 having different and distinguishable masses and / or charges. In some examples, an oligonucleotide may anneal to a second oligonucleotide and the second oligonucleotide may anneal to a third oligonucleotide, such as illustrated by the conjugate 800 and the oligonucleotide 808-1 annealing to second oligonucleotide 812-1 which anneals to the third oligonucleotide 820.

[0138] In any of the examples, a plurality of complexes may be formed using a plurality of conjugates, such as those provided in a kit. As an example, the plurality of protein-oligonucleotide complexes may include respective oligonucleotides, such as oligonucleotide 808-1 , 808-2, 808-3, 808-4, which are each different and that are designed to anneal to different second oligonucleotides (e.g., 812-1 , 812-2, 812-3, 812-4) to may cause each formed complex (e.g., 884-1 , 884-2, 884-3, 884-4) to have a different length from the others of the plurality and to provide separation for a plurality of target analytes. In some examples, each of the plurality of oligonucleotides (or at least a subset of the plurality) may be single and / or double stranded but of different lengths to provide a change in charge and / or mass that is sufficiently distinguishable from other complexes formed. This change in charge and / or mass may be used to derive an output signal and / or used to perform electrophoretic separation. In other examples and / or in addition, each of the plurality of oligonucleotides (or at least a subset of the plurality) may be single stranded and may have associated complementary single stranded second oligonucleotides which have different lengths from other second oligonucleotides to provide the change in charge and / or mass. In some examples, the second oligonucleotides may include modified nucleotides which are charge neutral, as described above. In some examples, the plurality of oligonucleotides (or subsets thereof) may additionally have varied lengths and / or third oligonucleotides with varied lengths may be used. In some examples, such as illustrated by conjugate 800, a particle and / or fluorophore may be used to enhance the signal. For example, the particleand / or fluorophore may be used to further change the charge and / or mass. In some examples, to further change the mass of the complex 884-1 , 884-2, 884- 3, 884-4 (e.g., target analyte + composition + second oligonucleotide) an additional signal component may be added.

[0139] In some examples, electrophoretic separation may be performed iteratively or across multiple cycles. For example, a first cycle of electrophoretic separation may be used to provide a crude separation to separate complexes into two or more groups, and then a second cycle (or more) used to separate the two or more groups into finer groups. Using the example modulation technique illustrated by the complex 884-1 , in some examples, the second and / or third oligonucleotides 812-1 , 820 may be added in different cycles to provide the iterative modulation in charge and / or mass. For example, the protein-oligonucleotide conjugate 800 that includes the protein 802-1 , tet 804-1 , TCO 806-1 , and oligonucleotide 808-1 may be exposed to a sample in a first cycle and binds to the target analyte 809-1 . After the first cycle of electrophoretic separation, the second oligonucleotide 812-1 (and optionally the third oligonucleotide 820) may be added to provide further separation in a second cycle of electrophoretic separation. If the second cycle does not provide sufficient separation, in some examples, the third oligonucleotide 820 may be added in a third cycle of electrophoretic separation. Examples are not limited to three cycles and may include addition of further signal components, such as additional oligonucleotides, detectable labels, particles, or other components and / or in further cycles.

[0140] FIG. 9 illustrates an example method of using a protein-oligonucleotide composition. The method 900 may be implemented using any of the compositions, protein-oligonucleotide conjugates, modulation techniques, and / or kits as previously described in connection with any of FIGs. 1-8.

[0141] As shown at 913, the method 900 includes exposing a sample to: (i) a protein; and (ii) an oligonucleotide linked to the protein via a tet and a TCO in a predetermined relationship. At 915, the method 900 includes identifying and / or using a complex formed that includes the protein or the oligonucleotide bound to the target analyte and the protein being linked to the oligonucleotide via the tetand TCO. In some examples, a plurality of complexes may be formed, which may include a plurality of the same complex and / or different complexes, such as a plurality of each of a plurality of different complexes formed using a set of target analytes.

[0142] In some examples, exposing the sample includes exposing the protein (linked to the tet) to the sample, wherein the protein or oligonucleotide is configured to bind to a target analyte. In such examples, the method 900 may further include removing unbound protein and oligonucleotide.

[0143] In some examples, identifying and / or using complexes formed further includes exposing the conjugates to signal constructs including a second oligonucleotide linked to a signal component via a linker, wherein the second oligonucleotide is complementary to the oligonucleotide. In some such examples, the method 900 includes at least one of: (i) exposing the sample to a volume of the protein linked to the oligonucleotide, wherein the protein or the oligonucleotide is configured to bind to a target analyte; (ii) removing protein of the volume that is unlinked to the target analyte; (iii) exposing the sample to a volume of signal constructs; (iv) removing second oligonucleotide unlinked to the target analyte; and (v) identifying the complexes formed by detecting an output signal associated with the signal constructs. In some such examples, the method 900 may further include removing or inactivating the signal component of the signal constructs. In some examples, removing unlinked protein and oligonucleotide may be performed using one washing step or multiple washing steps, as previously described.

[0144] In some examples, the method may include exposing the sample to a volume of the protein linked to the oligonucleotide (e.g., a PS) and identifying complexes formed by detecting an output signal associated with target analytes bound to the protein or the oligonucleotide. The output signal may be derived from the complex having a different charge and / or mass than the target analyte alone. In some examples, the protein-oligonucleotide composition may be used to modulate the charge and / or mass of the target analyte, such that the complex has a distinguishable charge and / or mass from another target analyte or from another complex formed with the other target analyte. For example, the PS maybe single or double stranded and acts as a signal construct. In other examples, the PS is single stranded or a hybrid of single stranded and double stranded, and the method further includes exposing the sample to a volume of signal constructs, where the signal constructs include at least a second oligonucleotide (e.g., a CS), and optionally a third oligonucleotide or other signal components (e.g., detectable label, particle).

[0145] In any of the above described examples, the signal construct may be set based on the anticipated concentration of the target analyte. For example, for a target analyte having a relatively low anticipated concentration, a detectable label of higher brightness may be used as compared to a target analyte having a relatively high anticipated concentration.

[0146] Various examples are directed to methods, computer-readable medium, and systems for designing a reagent-application (e.g., test or other type of use application) using at least one composition comprising a protein and an oligonucleotide. In various examples, a user interface may be used to guide a user through the design and allow for user control. The results may be output from the user computing device to the manufacturer for forming a set of protein- oligonucleotide conjugates, and other reagents (e.g., IS) for a particular reagent-application. The user interface may provide different displays depending on the particular type of reagent-application. As an example, the length of the DS, PS, CS and / or IS may be fixed or within a range for particular imaging reagent-applications and with each oligonucleotide being distinct from each other to either provide the different detectable labels or to bind to a different complementary IS, which may be set by the user selecting the particular application. As another example, with a proximity assay, the oligonucleotides may be distinct from each other with complementary end sequences. For immunoPCR, the oligonucleotides may be distinct from each other. With mobility modulation, such as for electrophoretic separation, the length of the oligonucleotides (e.g., PS, CS, or third oligonucleotide) may be modified to effect the change in charge and / or mass, as previously described. Additionally, the proteins or target analytes may be selected and the number of available wavelengths or other detectable signals may be selected and / orentered. Based on the user inputs, the user interface may display a plurality of potential proteins or aptamers associated with a type of reagent-application for supplying.

[0147] FIG. 10A-10D illustrate example devices including non-transitory computer-readable medium storing executable instructions. The devices 1000, 1001 , 1003, 1005 may include a computing device, such as a computer.

[0148] In some examples, each device 1000, 1001 , 1003, 1005 includes processing circuitry 1050, 1051 , 1070, 1071 and computer-readable medium 1060, 1061 , 1080, 1081 storing a set of instructions 1062, 1063, 1064, 1065, 1066, 1067, 1082, 1083, 1084, 1085, 1086, 1087. The computer-readable medium 1060, 1061 , 1080, 1081 may, for example, include read-only memory (ROM), random-access memory (RAM), electrically erasable programmable read-only memory (EEPROM), Flash memory, a solid state drive, and / or discrete data register sets. While illustrated on a single device 1000, 1001 , 1003, 1005, the set of instructions 1062, 1063, 1064, 1065, 1066, 1067, 1082, 1083, 1084, 1085, 1086, 1087may be stored on one or a plurality of non- transitory computer-readable medium and executed by one or more processor circuits, such a single computing device or distribution across multiple computing devices, such as with cloud computing. In some examples, a single device may execute different combinations of the different sets of instructions 1062, 1063, 1064, 1065, 1066, 1067, 1082, 1083, 1084, 1085, 1086, 1087 to iteratively guide a user for designing different types of analyses.

[0149] As shown by FIG. 10A, in some examples, the processing circuitry 1050 executes instructions 1062 to provide a user interface including a display of a plurality of types of reagent-applications. The processing circuitry 1050 executes instructions 1064, in response to user input selecting a reagentapplication of the plurality of types of reagent-applications, to revise the user interface to display a plurality of sub-types of the selected type of reagentapplication. The processing circuitry 1050 may further execute instructions 1066, in response to user input indicating the sub-type of the plurality of subtypes, to further revise the user interface to display a plurality of potential proteins or aptamers associated with the selected sub-type.

[0150] In some examples, the plurality of sub-types includes different targets for the selected type of reagent-application. For example, different targets may include different types of cancer, specific disease or infection, bacteria, among other diseases and pathogens. In some examples, the targets may be small molecules. In some examples, the display of the plurality of sub-types includes selectable icons, dropdown boxes, and / or an input field. For example, the different selectable icons, dropdown boxes, and / or an input field may include a predetermined set of target types and / or an input field for a user to input the sub-type. In some examples, the plurality of sub-types may include a specific type of the reagent-application, as further described herein.

[0151] In some examples, the display of the plurality of potential proteins or aptamers associated with the selected sub-type are automatically populated by the processing circuitry 1050 using data indicative of available proteins or aptamers configured to bind to a target analyte associated with the selected sub-type of the selected type of reagent-application. For example, the device 1000 may include or may be in communication with a device having a database containing target analytes known to be associated with different sub-types of reagent-applications. As an example, the database may include different types of cancers and target analytes associated with the different types of cancers as well as proteins or aptamers configured to bind to the associated target analytes. Examples are not limited to cancer, and may include other types of target analytes, including but not limited to different types of diseases and pathogens. In some such examples, the display of the plurality of potential proteins or aptamers further includes an input field for a user to input a protein or aptamer not included in the plurality of potential proteins or aptamers which are automatically populated.

[0152] In some examples, as noted above, the display of the plurality of potential proteins or aptamers includes an input field for a user to input a protein or aptamer. For example, a plurality of potential proteins or aptamers may be displayed along with an input field for a user to input another protein or aptamer that is not part of the plurality. In some examples, responsive to user input indicating the protein or aptamer, the processing circuitry 1050 may executefurther instructions to store data indicating the protein or aptamer is associated with the sub-type of the selected type of reagent-application, such as updating the data (e.g., update the database).

[0153] In some examples, responsive to user input selecting a set of proteins or aptamers from the plurality of potential proteins or aptamers and / or as manually input, the processing circuitry 1050 may execute further instructions to revise the user interface to display the selected set of proteins or aptamers with oligonucleotides. In some examples, additional user input may be provided, such as input to select the oligonucleotide length, the oligonucleotide being single and / or double stranded, second oligonucleotides, and / or selection of signals (e.g., detectable label or other types of signals) for a signal construct, among other variations, at least some of which are illustrated herein.

[0154] In some examples, the user may select from the plurality of types of reagent-applications, and upon making the selection via user input, the display of the user interface is revised to show pre-tested options (e.g., panels of varying protein and oligonucleotide combinations that are predefined, i.e. , have been tested for the specific reagent-application) to choose from, which may be in addition to creating a custom experimental tool kit for the user. Panels may be displayed by the number of channels available to the user (e.g., the frequencies of these channels), the total quantity of protein-oligonucleotide conjugates the user wants (e.g., to purchase), among other criteria. The experimental tool kit option may allow for the user to select specific proteins and / or aptamers and configurable oligonucleotides, and signal constructs, with examples further shown herein by FIGs. 11A-14C. In some examples, the user may only have one or two items to select from on the user interface, before moving on to next order decision, so as to minimize the confusion of developing their experimental tool kit.

[0155] As an example, a method of ordering protein-oligonucleotide conjugates or other compositions may include the processing circuitry 1050 executing instructions 1062 to provide a user interface including a display with of the plurality of types of reagent-applications for the user to select. In response to the user selecting one of the reagent-applications in the user interface (e.g.,via user input), revising the user interface to display with the options to select a pre-tested kit, an experimental tool kit, or a combination for purchasing. In response to the user selecting one of the options, and if an experimental tool kit or a combination is selected, in response to prompting the user through options (as further described herein), the processing circuitry 1050 (automatically) provides an output to the manufacturer (e.g., to other processing circuitry) indicating the type of reagent-application, the type of kit, and the components for the kit for generating the kit for the user.

[0156] In some examples, the user interface may include additional variations, such as prompts for the user to select how the protein-oligonucleotide conjugates or other compositions are delivered. For example, the user may select whether the protein-oligonucleotide conjugates or other compositions are delivered lyophilized or in a carrier. If a carrier is selected, the user may select to use a sacrificial protein, such as Bovine serum albumin (BSA), in the carrier, which may be used for enzyme-linked immunosorbent assay (ELSA) or other purposes.

[0157] In some examples, the user interface may include display(s) inquiring how many oligonucleotides to connect together and spacing requirements, such as for a SAPO multimer. For example, the user may select or otherwise provide user input to the user interface indicating the number of oligonucleotides to form the SAPO multimer and spacing requirement(s). The user interface may be revised to display options (e.g., suggestions) of CS and PS pairs that satisfy the input provided by the user, such as described further by FIG. 10C.

[0158] FIG. 10B illustrates a device 1001 as described in connection with FIG. 10A, with additional or alterative instructions 1063, 1065, 1067.

[0159] As shown by FIG. 10B, in some examples, the processing circuitry 1051 executes instructions 1063 to set a number of cycles for performing a reagent-application on a sample based on user input indicative of a number of proteins for the reagent-application and a number of signals for the reagentapplication. In various examples, the number of cycles is dependent on the number of signals and the number of proteins, as further illustrated herein. The processing circuitry 1051 may execute instructions 1065 to present a userinterface including a display of the set number of cycles with the number of signals for each of the number of cycles. In some examples, the display may be further based on a wavelength emission detection of imaging equipment to be used for the reagent-application and filters available.

[0160] As further illustrated by FIG. 12A, the display may include a table which includes the set number of cycles and the available channels (which are associated with the signals) for each cycle, and which may allow for user input to the device 1001 to select which protein are in which cycle and associated with a respective one of the available channels. In some examples, the processing circuitry 1051 may automatically populate the table by (randomly or selectively) assigning each protein to one cycle and to one of the channels, which may be modified by the user. As further illustrated by FIG. 12A, in response to user input, the processing circuitry 1051 may execute instructions 1067 to assign the number of proteins to one of the number of cycles and one of the number of signals (associated with one channel). In some examples, the processing circuitry 1051 may further execute instructions to revise the user interface to display the assignment in response to the user input.

[0161] In some examples, the user input may further indicate a type of reagent-application, which may indicate the type of oligonucleotides (e.g., length, single / double bonded (or both), DNA / RNA (or both), among other differences) and / or proteins to use. For example, the instructions may be executable by the processing circuitry 1051 to identify a plurality of potential proteins responsive to received user input indicative of the type of reagentapplication, and the display of the user interface may further include the plurality of potential proteins for selecting by the user as displayed on a display screen of the device 1001 . In some such examples, the user interface may further include a display of an input field for entering manual proteins (e.g., spaces in which text may be entered, dropdown boxes with proteins that may be selected, among other text and / or graphics) as described above.

[0162] FIG. 10C illustrates a device 1003 as described in connection with FIG. 10A, with additional or alterative instructions 1082, 1084, 1086.

[0163] As shown by FIG. 10C, in some examples, the processing circuitry 1070 executes instructions 1082 to provide a user interface including a display of a plurality of potential proteins associated a mobility modulation reagentapplication of a target. The processing circuitry 1070 may execute instructions 1084, in response to user input to select a number of the plurality of potential proteins, to revise the user interface to display indications for user input to select: a length of the oligonucleotides to be linked to the selected number of the plurality of potential proteins; and the oligonucleotides being single stranded, double stranded, or a combination thereof. The processing circuitry1070 may execute instructions 1086 to, in response to the user selection of the length of the oligonucleotides and being single and / or double stranded, assign the proteins to oligonucleotides and signal constructs. In some examples, the processing circuitry 1070 further executes the instructions to revise the user interface to display the assignment in response to user input.

[0164] In some examples, the processing circuitry 1070 further executes instructions to revise the user interface to display indications for user selection of: (i) oligonucleotides to bind to the selected ones of the plurality of potential proteins; (ii) the oligonucleotides being DNA and / or RNA; (iii) a length of second oligonucleotides which are respectively complementary to one of the oligonucleotides; (iv) wavelength, fluorophore emission, and / or fluorophore excitation; (v) the second oligonucleotides; and / or (vi) mass or charge change associated with complexes formed by proteins linked to oligonucleotides which bind to target analytes associated with the target, as well as various combinations thereof.

[0165] FIG. 10D illustrates a device 1005 as described in connection with FIG. 10A, with additional or alterative instructions 1082, 1084, 1086.

[0166] As shown by FIG. 10D, in some examples, the processing circuitry1071 executes instructions 1083 to provide a user interface including a display of a plurality of potential proteins associated an imaging reagent-application of a target. The processing circuitry 1071 executes instructions 1085, in response to user input to select a number of the plurality of potential proteins, to revise the user interface to display indications for user input to select: (i) a wavelength; (ii)fluorophore emission; (iii) fluorophore excitation; (iv) a length of the oligonucleotides to be linked to the selected number of the plurality of potential proteins; and / or (v) a length of second oligonucleotides which are respectively complementary to one of the oligonucleotides. The processing circuitry 1071 executes instructions 1087, in response to the user selection of the length of the oligonucleotides and being single and / or double stranded, to assign the selected number of proteins to an oligonucleotide of the selected oligonucleotides, a second oligonucleotide of the selected second oligonucleotides and a signal construct associated with the selected wavelength, fluorophore emission, and fluorophore excitation. In some examples, the processing circuitry 1071 further executes the instructions to revise the user interface to display the assignment in response to user input. |00167| In any of the described examples of FIGs. 10A-10D, the revisions to the user interface include a set of sequential displays to provide user input for each selection, selection in one display causing the next display to be provided by any of the processing circuitry 1050, 1051 , 1070, 1071. The sequential display may be used to guide the user through the design of reagents for a reagentapplication in a convenient and efficient manner.

[0168] In any of the examples, the type of reagent-application may determine a length range for the oligonucleotide and / or second oligonucleotide. For example, different types of reagent-applications may be associated with the use of oligonucleotide(s) of a length of between 30 nucleotides and about 65 nucleotides, 30 nucleotides and about 70 nucleotides, 20 nucleotides and about 80 nucleotides, 5 nucleotides and about 100 nucleotides, and / or 5 nucleotides and about 500 nucleotides, as further described below.

[0169] FIGs. 11A-11 B illustrate an example user interface including displays which may be provided by the device of FIG. 10A. As shown by FIGs. 11A-11 B (as well as FIGs. 12A-14C), the user interface 1110 may include a display of a variety of different options and / or selections for designing a reagent-application (e.g., which is sometimes herein interchangeably referred to as “use”, such as illustrated in FIGs. 11A, 12A,13A, and 14A) and which may be displayed on adisplay screen of a computing device accessible by a user for providing user inputs.

[0170] A user interface may include a viewable area or space, which is displayed on a display screen of a device, which provides for user-device interaction and communication. The user interface may include the display provided on a display screen of the device (e.g., screen of a monitor and / or a mobile device), on which text and / or graphics are displayed to the user and which the user may provide user inputs using a keyboard, a mouse, a touchpad, a touchscreen, or other input sources. In some examples, the user interface may include a graphic user interface.

[0171] In some examples, a user may log into a program and / or a computer application (e.g., app) for designing a reagent-application, which may be provided by a provider and / or manufacturer of reagents. For example, the user may input a username and password to log into the program. The design of the reagent-application may be used to set the reagents used for performing the reagent-application, where at least some of the reagents may include an implementation of and / or be provided as any of the compositions, protein- oligonucleotide conjugates, and / or kits as described in connection with any of FIGs. 1-8, 16A-18, and / or used to implement the method as described in connection with FIG. 9. In some examples, the user interface 1110 of FIG. 11A and / or FIG. 11 B may be implemented by any of device 1000, 1001 , 1003, 1005 of FIGs. 10A-10D.

[0172] In accordance with various examples, the user input to the user interface may dynamically adjust the displays provided on a display screen to aid a user in choices for designing the reagent-application and which is used to design and form the reagents. In many instances, reagents are expensive and / or time consuming to obtain, and the dynamic adjustment of the display that guides the user may reduce the risk of ordering incorrect reagents or reagents that may not work for the reagent-application. As the device(s) described herein may be used to design protein-oligonucleotide conjugates for analysis and research work, the user interface(s) may be used for customizing and creating unique combinations. In some examples, respective protein-oligonucleotides(and optional CS) may be pre-formed by the provider. Such protein- oligonucleotides may include known proteins and / or oligonucleotides that are configured to bind to specific targets, with respective having been tested to ensure the of protein-oligonucleotides do not cross-react with others in a set, among other assessments. As further illustrated herein, the user interface may display the respective potential proteins and / or oligonucleotides as a list and / or for an automatic options, with the display highlighting proteins and / or oligonucleotides which are part of the set of preformed protein-oligonucleotides (e.g., bold or highlighted text, listing first, etc.). For example, sub-portions of the plurality of potential proteins or aptamers or the oligonucleotide(s) displayed by the user interface for selection may include proteins and / or oligonucleotides from the set of preformed protein-oligonucleotides, and which are highlighted from others in the plurality.

[0173] As shown by FIG. 11 A, the user interface 1110 may include displays 1 , 2, 3 which are revised responsive to user inputs. The different displays 1 , 2, 3 may be provided in different sequential orders, and are not limited to that illustrated by FIG. 11 A (and / or FIG. 11 B).

[0174] After logging in, the user may select to design a new reagentapplication, and in response the user interface 1110 may be provided, which includes the display at 1 , including text and / or graphic(s) for selecting a type of reagent-application. The text or graphic may include an input field for the user to provide the text, a dropdown box with predefined reagent-applications available, selectable icons of predefined reagent-applications available, or other types of text and / or graphics.

[0175] In response to the user providing input to the device to select the type of reagent-application, the user interface 1110 may be revised to provide the display at 2, which includes text and / or graphic(s) for selecting a sub-type of the selected reagent application. In some examples, the display at 2 may include multiple revised displays as shown by 2A, 2B, 2C. For example, the selected reagent-application may include different sub-types. In some examples, the subtypes may be separately displayed at 1 . In some examples, a pop-up box or other revised display is provided at 2A. For example, different sub-types of animaging reagent-application may include Multiplex Immunofluorescence, e. g., Cyclic Immunofluorescence, Single Channel Immunofluorescence, and FRET, among others. Different sub-types for mobility modulation may include modulating charge and modulating mass, among others.

[0176] In some examples, alternatively or in addition, the sub-types may be associated with particular targets and / or sample types (e.g., tissue, serum, saliva). For example, the user interface 1110 may be revised to provide the display at 2B, which includes text and / or graphic(s) for selecting a target for the selected reagent-application. Example targets include cancer, virus, bacteria or other pathogens, proteins, molecules, cell, nucleic, acid and others. In some examples, the user may manually enter the target in an input field. In some examples, the target(s) may be provided as selectable icons and / or in dropdown box, among other types of text and / or graphics. In some examples, the user interface 1110 may be revised to provide the display at 2C, which includes text and / or graphic(s) indicating the user is to select a subtype of the selected target, such as a specific type of cancer (e.g., breast cancer, small cell lung cancer, brain cancer), a specific virus, bacteria or other pathogen, etc.

[0177] In response to the user providing input to the device to select the subtype of the selected reagent application, the user interface 1110 may be revised to provide the display at 3, which includes text and / or graphic(s) for selecting proteins. The text and / or graphics(s) may include input fields for the user to manually enter the proteins and / or a plurality of potential proteins available to the user, which may be provided as dropdown boxes, different selectable icons, and / or otherwise.

[0178] The user interface 1110 may include variations including different displays and orders of displays. FIG. 11 B illustrates some example variations of the user interface 1110 including different displays which may be provided in addition to those illustrated by FIG. 11 A. In some examples, any of the user interfaces shown herein may include additional displays and / or orders. For example, to design a multimer, the specific linkers, and / or first and second oligonucleotides (e.g., PS and secondary strands), among other components, may be displayed. Additional user interface displays (or in addition to thedisplays illustrated) may include indications to select for the number of CS, relative position(s) of the CS(s) to PS, and / or other placement criteria.

[0179] In some examples, as shown by FIG. 11 B, the user interface 1110 may be revised to provide the display at 2C, which includes text and / or graphic(s) for selecting a target fluorophore or other type of detectable label. In some examples, the selection may be based on a concentration level of target analyte(s). In other examples or in addition, and as further illustrated by FIG. 12B, the selection of the fluorophore or other detectable label may include a direct user selection. The text and / or graphics may include different selectable icons (e.g., each including a different fluorophore), an input field for which the user may input the fluorophore(s), and / or a dropdown boxes to select the fluorophores, among other types of text and / or graphics. In some examples, the user may select a “custom icon” which causes the user interface 1110 to be revised with a pop-up box over the display at 2C, with the pop-up box including an input field for the user to input the fluorophore, select an emission and / or excitation wavelength, among other text and / or graphics.

[0180] In some examples, as shown by FIG. 11 B, the user interface 1110 may be revised to provide the display at 4, which includes text and / or graphic(s) for selecting an oligonucleotide. The selection may be for a PS, a DS, an IS, a CS, or other oligonucleotides (e.g., third oligonucleotide), indicating single stranded and / or double stranded, indicating RNA and / or DNA, and / or inputting custom oligonucleotide sequences. The text and / or graphics may include different selectable icons, input field(s) for which the user may input the oligonucleotide(s) or properties thereof, and / or a dropdown boxes, among other types of text and / or graphics.

[0181] In some examples, as shown by FIG. 11 B, the user interface 1110 may be revised to provide the display at 5, which includes text and / or graphic(s) for selecting signal constructs. The selection may include types of signals and / or modulation used to derive the signal (e.g., wavelengths, channels, mass v. charge, emission excitation). The text and / or graphics may include different selectable icons, an input field for which the user may input the signal constructor properties thereof, and / or a dropdown boxes, among other types of text and / or graphics.

[0182] In some examples, as shown by FIG. 11 B, the user interface 1110 may be revised to provide the display at 6, which includes text and / or graphic(s) for selecting the predetermined relation of the protein to tet. The selection may be based on inputs provided by the user, such as those input in response to the displays at 1-3 of FIG. 11A. The text and / or graphics may include different selectable icons, an input field for which the user may input the signal construct or properties thereof, such as illustrated by the “custom icon”, and / or a dropdown boxes, among other types of text and / or graphics. In some examples, the selection options may include an auto option, in which the relationship is set based on prior response from the user. As an example, the default may be 1 :1 , with a particular reagent-application benefiting from a different relationship. For example, some targets may be present in low concentration in samples or specific target analytes associated therewith may have low concentrations.Such targets may benefit from signal amplification, which may be provided by having a predetermined relationship of p:1 ,1 :n, p:n, 1 :o, p:o, and / or using multimers.

[0183] In some examples, the length of the oligonucleotide(s) used may be dependent on the reagent-application selection. For example, for proximity assays, oligonucleotides may be used that are 30 to 65 nucleotides long, 20 to 80 nucleotides long, or 5 to 100 nucleotides long. For ImmunoPCR having oligonucleotides that are distinct from one another, oligonucleotides may be used that are 30 to 70 nucleotides long, 20 to 80 nucleotides long, or 5 to 100 nucleotides long. For mobility modulation, oligonucleotides may be used that are 5 to 500 nucleotides long. Examples are not so limited and may include other variations.

[0184] In some examples, in response to the user going through the displays of the user interface 1110, reagents for the reagent-application are designed for ordering. For example, the user interface 1110 may be revised to provide the display at 7, which is associated with ordering the reagents and which may include text and / or graphic(s) showing the reagents, including the proteins,oligonucleotides, and optionally signal constructs. The text and / or graphics may show a set of reagents designed to perform a reagent-application associated with a set of target analytes. The text or graphics may allow for the user to manually revise the same and / or to order the reagents. In some examples, the user interface 1110 may allow the user to save the design and / or to order the reagent-application from a provider (e.g., manufacturer). For example, the user interface 1110 may include a display including a selectable icon for the user to select to send to the provider for providing an estimated cost to form the reagents for the reagent-application and / or to form the reagents for the reagentapplication which may include a plurality of protein-oligonucleotide conjugates, and optionally, second oligonucleotides and / or the set of signal constructs. The cost of readily available components (e.g., preformed protein-oligonucleotides) being integrated into a protein-oligonucleotide composition may not require a quote and may be displayed to the user for a direct purchase.

[0185] FIGs. 12A-12B illustrate an example user interface including displays which may be provided by the device of FIG. 10B. As shown by FIG. 12A, the user interface 1210 may include displays 1 , 2, 3A, 3B, 4 which are revised responsive to user inputs. The different displays 1 , 2, 3A, 3B, 4 may be provided in different sequential orders, and are not limited to that illustrated by FIG. 12A (and / or FIG. 12B).

[0186] As with FIGs. 11A-11 B, the user may log into the program and / or computer-application to design a reagent-application. After logging in, the user may select to design a new reagent-application, and in response the user interface 1210 may be provided, which includes the display at 1 , including text and / or graphic(s) for selecting a type of reagent-application. The text or graphic may include an input field for the user to provide the text, a dropdown box with predefined reagent-applications available, selectable icons of predefined reagent-applications available, or other types of text and / or graphics.

[0187] In response to the user providing user input to the device to select the type of reagent-application of CycIF, the user interface 1210 may be revised to provide the display at 2, which includes text and / or graphic(s) for entering or selecting proteins. The text and / or graphics(s) may include input fields for theuser to manually enter the proteins and / or a plurality of potential proteins available to the user, which may be provided as dropdown boxes, different selectable icons, and / or otherwise.

[0188] In response to user input selecting the set of proteins, the user interface 1110 may revised to provide the display at 3A, which includes text and / or graphic(s) for entering (or otherwise selecting) the number of wavelengths to use for the reagent-application. The number of wavelengths may be limited by the type of imaging circuitry used (e.g., microscope) and / or the type of reagentapplication. The text and / or graphics may include an input field for which the user may input a number, different selectable icons (each of a different number), and / or a dropdown box to select the number, among other types of text and / or graphics.

[0189] In response to the user selected the number of wavelengths available, in some examples, the user interface 1110 may be revised to provide the display at 3B, which includes text and / or graphic(s) showing the number of channels to use for the reagent-application and which are associated with the selected number of wavelength (e.g., equal to). The display at 3B may not include space for user input text and / or may alternatively include an icon for the user select to go the next display.

[0190] After a period of time and / or in response to the user selecting the icon, the user interface 1210 may be revised to provide the display at 4, which includes text and / or graphic(s) showing the number of cycles, the proteins to be analyzed in each cycle, and the channel (e.g., “CH.”) to use for each protein and in each cycle. In various examples, as previously described in connection with FIG. 10B, processing circuitry may determine the number of cycles to use for the reagent-application based on the number of proteins (which is indicative of the number of target analytes) and the number of channels, and then may provide the display at 4. In some examples, the processing circuitry may automatically populate the table by randomly or strategically assigning each protein to one cycle and to one of the channels. In some examples, the user may select (or revise the automatic assignment) the assignment. For example, the user may provide user input to select or revise which proteins are inrespective cycles and / or are associated with respective channels from the automatic assignment by the processing circuitry. While the various user interfaces, including user interface 1210, show the proteins being in alphabetical order (e.g., ABC), examples are not so limited. In some examples, respective proteins may be listed before others and / or otherwise highlighted, such as proteins which are within a set of preformed protein-oligonucleotides.

[0191] The user interface 1210 may include variations including different displays and orders of displays. For example, FIG. 12A illustrates an example of an imaging reagent-application. Other types of reagent-applications may be selected, which result in different types of displays and / or selections.

[0192] Additionally, different selections may be made using the user interface 1210, which may be optional. FIG. 12B illustrates some example variations of the user interface 1210 including different displays which may be provided in addition to those illustrated by FIG. 12A.

[0193] In some examples, as shown by FIG. 12B, the user interface 1210 may be revised to provide the display at 5A, which includes text and / or graphic(s) for selecting a target fluorophore or other type of detectable label. In some examples, the selection may be based on a concentration level of target analytes. The text and / or graphics may include different selectable icons (e.g., each including a different concentration level of the target analyte), an input field for which the user may include the concentration level of a target analyte, and / or a dropdown box to select the concentration level of a target analyte, among other types of text and / or graphics. While the display at 5A shows the concentration levels of target analytes as high, medium, and low, examples are not so limited and may include other texts or values, such as values from 1-5 or from 1-10. Similarly, the target concentration may be different for different target analytes within a sample. In some examples, the display at 5A may include text to indicate the user should select based on the lowest concentration of all target analytes and / or may allow for selection of the concentration for each target analyte.

[0194] In some examples, the selection of the fluorophore or other detectable label may include a direct user selection. For example, as shown by FIG. 12B,the user interface 1210 may be revised to provide the display at 5B, which includes text and / or graphic(s) for selecting fluorophores. The text and / or graphics may include different selectable icon (e.g., each including a different fluorophore), an input field for which the user may input the fluorophore(s), such as illustrated by the “custom icon”, and / or a dropdown boxes to select the fluorophores, among other types of text and / or graphics. In some examples, the user may select a “custom icon” which causes the user interface 1210 to be revised with a pop-up box over the display at 5B, with the pop-up box including an input field for the user to input the fluorophore.

[0195] In some examples, shown by FIG. 12B, the user interface 1210 may be revised to provide the display at 5C, which includes text and / or graphic(s) for selecting oligonucleotides. The text and / or graphics may include different selectable icons, an input field for which the user may input the oligonucleotide(s), such as illustrated by the “custom icon”, and / or a dropdown boxes to select the oligonucleotides, with each dropdown box including a plurality of potential (e.g., available) oligonucleotides (and once one is selected, it is not available for further selection), among other types of text and / or graphics. The number of oligonucleotides (e.g., Z) to select may be set based on the number of proteins entered and / or selected by the user, such as at the display at 2 of FIG. 12A. In some examples, the selectable icons or options in the dropdown boxes may include each of the plurality of potential oligonucleotides, an automatic option (e.g., AUTO), and / or a custom option. For example, the icon “AUTO” may be selected and in response, the device selects a set of Z oligonucleotides without further user input. As another example, the user may select the “custom icon” (or which may be an option in a dropdown box) which causes the user interface 1210 to be revised with a pop-up box over the display at 5C, with the pop-up box including an input field for the user to input a custom oligonucleotide. As previously described, the plurality of potential oligonucleotides may be dependent on the reagent-application selected (e.g., setting the length of the oligonucleotides) and on the number of proteins (e.g., setting the number of oligonucleotides).

[0196] In some examples, in response to the user going through the displays of the user interface 1210, reagents for the reagent-application are designed. As previously described, in some examples, the user interface 1210 may allow the user to save the design and / or to order the reagent-application from a provider (e.g., manufacturer). For example, the user interface 1210 may include a display including a selectable icon for the user to select to send to the provider for providing an estimated cost to form the reagents for the reagent-application and / or to form the reagents for the application which include a plurality of protein-oligonucleotide conjugates, and optionally, second oligonucleotides and / or the set of fluorophores.

[0197] FIGs. 13A-13C illustrate an example user interface including displays which may be provided by the device of FIG. 10C. As shown by FIG. 13A, the user interface 1310 may include displays 1 , 2, 3, 4, 5 ,6 which are revised responsive to user inputs. The different displays 1 , 2, 3, 4, 5, 6 may be provided in different sequential orders, and are not limited to that illustrated by FIG. 13A (and / or FIGs. 13B-13C).

[0198] As with FIGs. 11A-11 B, the user may log into the program and / or computer-application to design a reagent-application. After logging in, the user may select to design a new reagent-application, and in response the user interface 1310 may be provided, which includes the display at 1 , including text and / or graphic(s) for selecting a type of reagent-application. The text or graphic may include an input field for the user to provide the text, a dropdown box with predefined reagent-applications available, selectable icons of predefined reagent-applications available, or other types of text and / or graphics.

[0199] In response to the user providing user input to the device to select the type of reagent-application of mobility modulation, the user interface 1310 may be revised to provide the display at 2, which includes text and / or graphic(s) for entering or otherwise selecting proteins. The text and / or graphics(s) may include input fields for the user to manually enter the proteins and / or a plurality of potential proteins available to the user, which may be provided as dropdown boxes, different selectable icons, and / or otherwise, as previously described.

[0200] In response to user input selecting the set of proteins, the user interface 1310 may revised to provide the display at 3, which includes text and / or graphic(s) for selecting whether the PS is double stranded (e.g., DS in the figure) and / or single stranded (e.g., SS in the figure). The text and / or graphics may include an input field, different selectable icons, and / or dropdown boxes to select, among other types of text and / or graphics. In some examples, the selection may allow for a hybrid, as previously described.

[0201] In response to the user selecting the PS being double stranded and / or single stranded, in some examples, the user interface 1310 may be revised to provide the display at 4, which includes text and / or graphic(s) for selecting the length of the PS. The display at 4 may include an input field for which the user may input a number, different selectable icons (each of a different number), and / or dropdown boxes to select the number, among other types of text and / or graphics. In some examples, the selection may include a selectable icon “custom” which allows for the user to input any number and / or may include an automatic option, in which the processing circuitry selects the number, which may be based on the target analyte(s). In some examples, the PS may be used as the signal construct and may be varied. In some such examples, the display at 4 may provide for a range of PS length.

[0202] In response to the user selecting the PS length, in some examples, the user interface 1310 may be revised to provide the display at 5, which includes text and / or graphic(s) for selecting whether or not to use a CS. The display at 5 may include an input field, different selectable icons, and / or dropdown boxes, among other text and graphics.

[0203] In response to the user selecting whether to use a CS or not, the user interface 1310 may be revised to provide the display at 6, which includes text and / or graphic(s) showing the reagents including the proteins, associated PS and optional CS and the signal construct for each target analyte. In various examples, as previously described in connection with FIG. 10C, processing circuitry may assign each target analyte-protein to an oligonucleotide configuration and signal construct based on user inputs to the user interface 1310 and then may provide the display at 6. In some examples, the processingcircuitry may automatically populate the table by randomly or strategically assigning each target analyte-protein to an oligonucleotide configuration and signal construct. In some examples, the user may select (or revise the automatic assignment) the assignment, as previously described.

[0204] The user interface 1310 may include variations including different displays and orders of displays. For example, different selections may be made using the user interface 1310, which may be optional. FIGs. 13B-13C illustrate some example variations of the user interface 1310 including different displays which may be provided in addition to those illustrated by FIG. 13A.

[0205] In some examples, as shown by FIG. 13B, the user interface 1310 may be revised to provide the display at 1A, which includes text and / or graphic(s) for selection of a sub-type of a mobility modulation reagent-application. The different sub-types may include the modulation type, such as modulating charge, modulating mass, additional signal component, or others. In some examples, multiple may be selected. In some examples or in addition, the selection may include the selection of the modulation technique, such as varying the length of the PS, varying the length of the CS, varying the length of the PS and CS, using modified neutral charged nucleotides, using third oligonucleotides, using a detectable label or particle, among other variations and combinations thereof. The display at 1A may include input fields, selectable icons, and / or dropdown boxes, among other text and / or graphics.

[0206] In some examples, the user interface 1310 may be revised to select a detectable label to use as part of the signal construct, such as a fluorophore. For example, the user interface 1310 may be revised to provide the display at 1 B, which includes text and / or graphic(s) for selecting a wavelength to use. The user interface may further or alternatively be revised to provide the display at 1 C and 1 D, which respectively include text and / or graphs for selection of the fluorophore emission and fluorophore excitation. The text and / or graphics may include different selectable icons (e.g., each including different wavelengths, emissions, excitations), input fields, such as illustrated by the “custom icon”, and / or dropdown boxes, among other types of text and / or graphics.

[0207] In some examples, shown by FIG. 13C, the user interface 1310 may be revised to provide the display at 3A, which includes text and / or graphic(s) for selecting whether the PS is RNA and / or DNA. The text and / or graphics may include different selectable icons, an input field, and / or dropdown boxes, among others.

[0208] In some examples, the user interface 1310 may be further revised to provide displays to select the oligonucleotides. For example, the user interface 1310 may be revised to provide the display at 4A, which includes text and / or graphic(s) for selecting the PS. In addition, the user interface 1310 may be revised to provide the displays at 5A and 5B, which includes text and / or graphic(s) for respectively selecting a length of the CS and the CS. The text and / or graphics may include different selectable icons, an input field for which the user may input the oligonucleotide(s), such as illustrated by the “custom icon”, and / or dropdown boxes to select the oligonucleotides, with each dropdown box including one of a plurality of potential oligonucleotides (and once one is selected, it is not available for further selection), among other types of text and / or graphics. The number of oligonucleotides (e.g., Z) to select may be set based on the number of proteins entered and / or selected by the user, such as at the display at 2 of FIG. 13A. In some examples, the selectable icons or options in the dropdown boxes may include the plurality of potential oligonucleotides, an automatic option (e.g., AUTO), and / or a custom option. For example, the icon “AUTO” may be selected and in response, the device selects a set of Z oligonucleotides without further user input. As another example, the user may select the “custom icon” (or which may be an option in a dropdown box) which causes the user interface 1310 to be revised with a pop-up box over the display at 5B, with the pop-up box including an input field for the user to input a custom oligonucleotide. In some examples, the “custom” option may include the ability to indicate additional CS that are designed to hybridize to previously selected CS selections. As previously described, the plurality of potential oligonucleotides may be dependent on the reagent-application selected (e.g., setting the length of the oligonucleotides) and on the number of proteins (e.g., setting the number of oligonucleotides).

[0209] In some examples, the user interface 1310 may be further revised to provide displays indicating the user is to select the mobility modulation, such as selecting the mass component or charge component. For example, the user interface 1310 may be revised to provide the displays at 7A and / or 7B, which includes text and / or graphic(s) for selecting mass addition or charge addition. The text and / or graphics may include different selectable icons, an input field for which the user may input the addition(s), such as illustrated by the “custom icon”, and / or dropdown boxes, with each dropdown box including a set of additions, among other types of text and / or graphics. In some examples, the selectable icons or options in the dropdown boxes may include different signal component types, specific mass or charge to add or variations between complexes, an automatic option (e.g., AUTO), and / or a custom option. For example, the icon “AUTO” may be selected and in response, the device selects the mass or charge additions without further user input. As another example, the user may select the “custom icon” (or which may be an option in a dropdown box) which causes the user interface 1310 to be revised with a pop-up box over the display at 7A or 7B, with the pop-up box including an input field for the user to input a custom input.

[0210] In some examples, in response to the user going through the displays of the user interface 1310, reagents for the mobility modulation reagentapplication are designed. As previously described, in some examples, the user interface 1310 may allow the user to save the design and / or to order the reagent-application from a provider (e.g., manufacturer). For example, the user interface 1310 may include a display including a selectable icon for the user to select to send to the provider for providing an estimated cost to form the reagents for the reagent-application and / or to form the reagents for the application which include a plurality of protein-oligonucleotide conjugates, and optionally, second oligonucleotides and / or the set of fluorophores.

[0211] FIGs. 14A-14C illustrate an example user interface including displays which may be provided by the device of FIG. 10D. As shown by FIG. 14A, the user interface 1410 may include displays 1 , 2, 3, 4, 5, 6 which are revised responsive to user inputs. The different displays 1 , 2, 3, 4, 5, 6 may be providedin different sequential orders, and are not limited to that illustrated by FIG. 14A (and / or FIGs. 14B-14C).

[0212] As with FIGs. 11 A-11 B, the user may log into the program and / or computer-application to design a reagent-application. After logging in, the user may select to design a new reagent-application, and in response the user interface 1410 may be provided, which includes the display at 1 , including text and / or graphic(s) for selecting a type of reagent-application. The text or graphic may include an input field for the user to provide the text, dropdown boxes with predefined reagent-applications available, selectable icons of predefined reagent-applications available, or other types of text and / or graphics.

[0213] In response to the user providing user input to the device to select the type of reagent-application of imaging, the user interface 1410 may be revised to provide the display at 2, which includes text and / or graphic(s) for entering or otherwise selecting proteins. The text and / or graphics(s) may include input fields for the user to manually enter the proteins and / or a plurality of potential proteins available to the user, which may be provided as dropdown boxes, different selectable icons, and / or otherwise.

[0214] In response to user input selecting the set of proteins, the user interface 1410 may revised to provide the display at 3, which includes text and / or graphic(s) for selecting the wavelength(s) to use for the imaging reagentapplication. The wavelength used may be limited by the type of imaging circuitry used (e.g., microscope) and / or the type of reagent-application. The text and / or graphics may include an input field for which the user may input a number, different selectable icons (each of a different number), and / or dropdown boxes to select the number, among other types of text and / or graphics.

[0215] In response to the user selected the wavelength(s), in some examples, the user interface 1410 may be revised to provide the display at 4, which includes text and / or graphic(s) for selecting the PS length. The display at 4 may include an input field for which the user may input a number, different selectable icons (each of a different number), and / or dropdown boxes to select the number, among other types of text and / or graphics. In some examples, the selection may include a selectable icon “custom” which allows for the user toinput any number and / or may include an automatic option, in which the processing circuitry selects the number, which may be based on the target analyte(s).

[0216] In response to the user selecting the PS length, the user interface 1410 may be revised to provide the display at 5, which includes text and / or graphic(s) for selecting the CS length. The display at 5 may include an input field for which the user may input a number, different selectable icons (each of a different number), and / or dropdown boxes to select the number, among other types of text and / or graphics. In some examples, the selection may include a selectable icon “custom” which allows for the user to input any number and / or may include an automatic option, in which the processing circuitry selects the number.

[0217] In response to the user selecting the CS length, the user interface 1410 may be revised to provide the display at 6, which includes text and / or graphic(s) showing the reagents including the proteins, oligonucleotide configuration including the PS and CS, and the signal construct for each target analyte. In various examples, as previously described in connection with FIG. 10D, processing circuitry may assign each target analyte-protein to an oligonucleotide configuration and signal construct based on user inputs to the user interface 1410 and then may provide the display at 6. In some examples, the processing circuitry may automatically populate the table by randomly or strategically assigning each target analyte-protein to an oligonucleotide configuration and signal construct. In some examples, the user may select (or revise the automatic assignment) the assignment, as previously described.

[0218] The user interface 1410 may include variations including different displays and orders of displays. For example, different selections may be made using the user interface 1410, which may be optional. FIGs. 14B-14C illustrate some example variations of the user interface 1410 including different displays which may be provided in addition to those illustrated by FIG. 14A.

[0219] In some examples, the user interface 1410 may be revised to select a detectable label to use as part of the signal construct, such as selecting a fluorophore. For example, the user interface 1410 may be revised to provide thedisplays at 3A and 3B, which respectively include text and / or graphs for selecting the fluorophore emission and fluorophore excitation. The text and / or graphics may include different selectable icons (e.g., each including different wavelengths, emissions, excitations), input fields, such as illustrated by the “custom icon”, and / or dropdown boxes, among other types of text and / or graphics.

[0220] In some examples, the user interface 1410 may be further revised to provide displays to select the oligonucleotides. For example, the user interface 1410 may be revised to provide the display at 4A, which includes text and / or graphic(s) for selecting the PS. In addition, the user interface 1410 may be revised to provide the display at 5A, which includes text and / or graphic(s) for respectively selecting the CS. The text and / or graphics may include different selectable icons, input fields for which the user may input the oligonucleotide(s), such as illustrated by the “custom icon”, and / or dropdown boxes to select the oligonucleotides, with each dropdown box including a plurality of potential (e.g., available) oligonucleotides (and once one is selected, it is not available for further selection), among other types of text and / or graphics. The number of oligonucleotides (e.g., Z) to select may be set based on the number of proteins entered and / or selected by the user, such as at the display at 2 of FIG. 14A. In some examples, the selectable icons or options in the dropdown boxes may include the plurality of potential oligonucleotides, an automatic option (e.g., AUTO), and / or a custom option. For example, the icon “AUTO” may be selected and in response, the device selects a set of Z oligonucleotides without further user input. As another example, the user may select the “custom icon” (or which may be an option in a dropdown box) which causes the user interface 1410 to be revised with a pop-up box over the display at 4A and 5A, with the pop-up box including input field(s) for the user to input a custom oligonucleotide. As previously described, the plurality of potential oligonucleotides may be dependent on the reagent-application selected (e.g., setting the length of the oligonucleotides) and on the number of proteins (e.g., setting the number of oligonucleotides).

[0221] In some examples, as shown by FIG. 14B, the user interface 1410 may be revised to provide the display at 7, which includes text and / or graphic(s) for selecting whether signal amplification is to be used. The signal amplification may be provided by using multiple signal components (e.g., multiple fluorophores), the predetermined relationship, and / or multimers, such as illustrated by FIGs. 3C-3E and FIGs. 16A-18.

[0222] In some examples, the imaging reagent-application may include a particular sub-type of imaging. For example, as shown by FIG. 14C, the user interface 1410 may be revised to provide the display at 1A (and in response to selecting “imaging” at 1 ), which includes text and / or graphic(s) for selecting the sub-type of imaging reagent-application. The different sub-types may include FRET, CycIF, single channel, and other (or custom) options, among others.

[0223] Fluorescence Resonance Energy Transfer (FRET) is an imaging reagent-application involving the energy transfer between donor fluorophore and an acceptor fluorophore. The energy transfer between the donor and acceptor fluorophore is a function of the distance between the fluorophores, and may be used to determine a distance therebetween. In various examples, a FRET reagent-application may be used to a probe a distance or other relationship between two or more target analytes.

[0224] In some examples, in response to selecting FRET at 1A, as shown by FIG. 14C, the user interface 1410 may be revised to provide the display at 1 B, which includes text and / or graphic(s) for selecting the target analytes. The text and / or graphics may include different selectable icons (e.g., each including a different target analyte), input fields for which the user may input the target analytes, such as illustrated by the “custom icon”, and / or dropdown boxes to select the targets, among other types of text and / or graphics.

[0225] In response to the selection of the target analytes (and in alternative to display 2 of FIG. 14A), the user interface 1410 may be revised to iteratively provide the displays at 2A and 2B, which includes text and / or graphic(s) for entering or otherwise selecting proteins for the first target analyte and for the second target analyte. The text and / or graphics(s) may include input fields for the user to manually enter the proteins and / or a plurality of potential proteinsavailable to the user, which may be provided as dropdown boxes, different selectable icons, and / or otherwise.

[0226] In some examples, as shown by FIG. 14C, the user interface 1410 may be revised to provide the display at 8, which includes text and / or graphic(s) for selecting signal constructs. The text and / or graphics may include different selectable icons, input fields, such as illustrated by the “custom icon”, and / or a dropdown boxes, among other types of text and / or graphics. In some examples, the user may select a “custom icon” or “auto icon” which operates similar to that previously described. In some examples, the options for the signal constructs may include section of where the fluorophores are linked. For example, the fluorophores may be attached to each PS at the 5’ and / or 3’ ends, to one of the PS and to the CS at the 5’ and / or 3’ ends, and / or to one of the proteins and to the CS at the 5’ or 3’ end.

[0227] In some examples, shown by FIG. 14C, the user interface 1410 may be revised to provide the display at 6A (and which may be an implementation of the display at 6), which includes text and / or graphic(s) showing the reagents including the proteins, oligonucleotide configuration including the PS for each target analyte and a CS, and the signal construct for a FRET reagentapplication. As shown, for a FRET reagent-application, the oligonucleotide configuration may include a PS (e.g., PS1 , PS2) and protein (e.g., protein A, protein D) for each target analyte and a CS that is complementary to both PS. The signal construct may include two fluorophores and identification of what components the fluorophores are linked to.

[0228] In some examples, in response to the user going through the displays of the user interface 1410, reagents for the reagent-application are designed. As previously described, in some examples, the user interface 1410 may allow the user to save the design and / or to order the reagent-application from a provider (e.g., manufacturer). For example, the user interface 1410 may include a display including a selectable icon for the user to select to send to the provider for providing an estimated cost to form the reagents for the reagent-application and / or to form the reagents for the application which include a plurality ofprotein-oligonucleotide conjugates, and optionally, second oligonucleotides and / or the set of fluorophores.

[0229] FIGs. 15A-15C illustrate example protein-oligonucleotide conjugates or portions thereof. In some examples, two (or more) protein-oligonucleotide conjugates 1500-1 , 1500-2 may be used to perform a FRET reagent-application. Various features and attributes of each of the protein-oligonucleotide conjugates 1500-1 , 1500-2, the second oligonucleotides 1512, the signal constructs 1515, or portions thereof 1503, 1503 of FIGs. 15A-15C may include at least substantially the same features and attributes of the composition 100 of FIG. 1 and / or any of the compositions 200, 201 , 203 of FIGs. 2A-2D, as shown by the common numbering and with the details of the common features and attributes not being repeated. In some examples, the protein-oligonucleotide conjugates 1500-1 , 1500-2 or portions thereof of FIGs. 15A-15C may include an example implementation of the composition 100 of FIG. 1 and / or any of compositions 200, 201 , 203 of FIGs. 2A-2D.

[0230] As shown by FIG. 15A, in some examples, two protein-oligonucleotide conjugate 1500-1 , 1500-2 may be used which each include a respective protein 1502-1 , 1502-2, a tet 1504-1 , 1504-2, a TCO 1506-1 , 1506-2, and a single stranded oligonucleotide 1508-1 , 1508-2, which may each be referred to as a PS. In various examples, the proteins 1502-1 , 1502-2 are each configured to respectively bind to a different target analyte 1509-1 , 1509-2. In some examples, the protein-oligonucleotide conjugates 1500-1 , 1500-2 may be exposed to a sample. In response to the sample containing the target analytes 1509-1 , 1509-2, the first protein 1502-1 binds to the first target analyte 1509-1 and the second protein 1502-2 binds to the second target analyte 1509-2. A second oligonucleotide 1512 is added, which is complementary to both oligonucleotides 1508-1 , 1508-2 and may be referred to as a CS.

[0231] In various examples, two fluorophores 1514-1 , 1514-2 may be signal components that form part of a signal construct 1515. In some examples, as shown by FIG. 15A, the two fluorophores 1514-1 , 1514-2 may be respectively linked to the oligonucleotide 1508-1 , 1508-2 at the 5’ end and / or 3’ end. For example, the first fluorophore 1514-1 may be linked to the 5’ end of theoligonucleotide 1508-1 and the second fluorophore 1514-2 may be linked to the 3’ end of the oligonucleotide 1508-2. If the first and second target analytes 1509-1 , 1509-2 are present in the sample, the protein-oligonucleotide conjugates 1500-1 , 1500-2 bind thereto and the second oligonucleotide 1512 anneals to the oligonucleotides 1508-1 , 1508-2, which may cause the first and second fluorophores 1514-1 , 1514-2 to be within a threshold distance to another such that energy transfers between and a detectable signal is output. As may be appreciated, the detectable signal may be output only when the fluorophores 1514-1 , 1514-2 are within a threshold.

[0232] Examples are not limited to the fluorophores 1514-1 , 1514-2 being linked to each PS (e.g., 1508-1 , 1508-2). For example, FIG. 15B illustrates an example of the fluorophores 1514-1 , 1514-2 being linked to one of the PS and the other to the CS. In particular, FIG. 15B illustrates a sub-portion 1503 of the two protein-oligonucleotide conjugate 1500-1 , 1500-2 illustrated by FIG. 15A, but with the fluorophores being linked to the oligonucleotide 1508-1 and the second oligonucleotide 1512. In some examples, the second oligonucleotide 1512 may be pre-annealed to the oligonucleotide 1508-2. The sub-portion 1503 may be said to be a close-up view of the signal construct 1515 which includes the oligonucleotide 1508-1 , the second oligonucleotide 1512, and the fluorophores 1514-1 , 1514-2. The two protein-oligonucleotide conjugates 1500- 1 , 1500-2 may include the additional components illustrated by FIG. 15A, which are not illustrated by FIG. 15B for clarity purposes. In the example, the first fluorophore 1514-1 may be linked to the 3’ end of the oligonucleotide 1508-1 and the second fluorophore 1514-2 may be linked to the 5’ end of the second oligonucleotide 1512. Examples may include other variations, such as being linked to the oligonucleotide 1508-2 and being linked at other ends of the oligonucleotides 1508-1 , 1508-2, and / or 1512.

[0233] FIG. 15C illustrates an example of the fluorophores 1514-1 , 1514-2 being linked to one of the proteins and the other to the CS. In particular, FIG. 15C illustrates a sub-portion 1505 of the two protein-oligonucleotide conjugates 1500-1 , 1500-2 illustrated by FIG. 15A, but with the fluorophores being linked to the protein 1502-1 and the second oligonucleotide 1512. The sub-portion 1505may be said to be a close-up view of the signal construct 1515 which includes the protein 1502-1 , the second oligonucleotide 1512, and the fluorophores 1514-1 , 1514-2. The two protein-oligonucleotide conjugates 1500-1 , 1500-2 may include the additional components illustrated by FIG. 15A, which are not illustrated by FIG. 15C for clarity purposes. In the example, the first fluorophore 1514-1 may be linked to the protein 1502-1 which comprises an sdAb and the second fluorophore 1514-2 may be linked to the 5’ end of the second oligonucleotide 1512. As described above, in some examples, the second oligonucleotide 1512 may be pre-annealed to the oligonucleotide 1508-2. Examples may include other variations, such as being linked to the protein 1502-2 of FIG. 15A and being linked at other ends of the oligonucleotides 1508- 1 , 1508-2, and / or 1512.

[0234] In some examples, the FRET reagent-application may include an iterative process which includes attaching different length CS (e.g., second oligonucleotide 1512). By attaching different length CS, the length between the target analytes 1509-1 , 1509-2 may be probed. For example different length CS may be iteratively added, the sample is imaged to detect for the detectable signal, and then the particular CS is removed. The process may be repeated for a plurality of different length CS and to identify when the detectable signal is no longer changed.

[0235] FIGs. 16A-16G illustrate further example compositions comprising a protein and an oligonucleotide. In some examples, the compositions of FIGS. 16A-16G comprise and / or may be used to form multimers, as previously described above.

[0236] For example, FIG. 16A illustrates an example composition 1640 which is a multimer comprising a first protein 1602-1 , a second protein 1602-2, an oligonucleotide 1608, and a multimer linker 1630. The first and second proteins 1602-1 , 1602-2 are linked to the oligonucleotide 1608 via the multimer linker 1630. Examples are not limited to two proteins and a single oligonucleotide as shown by FIG. 16A. For example, various compositions may include additional proteins and / or additional oligonucleotides, such as more than two proteins. Aspreviously described above, the first and second proteins 1602-1 , 1602-2 may be the same or different protein from another.

[0237] A multimer linker refers to or includes a linker configured to bind more than two components, such as more than two protein(s) and / or oligonucleotide(s). For example, the multimer linker may form part of a multimer. Various different multimer linkers may be used. Some specific and non-limiting example multimer linkers include amine-PEG4-amide-Tri(3- methoxypropanamide-PEG10-Propargyl)Methane HCI salt, (Methyltetrazine- PEG10)-Tri-(Azide-PEG10-ethoxymethyl)-methane, Amino-Tri-(Azide-PEG4- ethoxymethyl)-methane, Acid-PEG25-Amide-Tri(3-methoxypropanamide- PEG23-Azide) Methane, and DBCO-PEG4-Amido-tri(PEG6-TCO)-methane, among other linkers and modified forms thereof. In some examples, the multimer linker 1630 includes branches (e.g., arms) which may be used to link multiple proteins and / or multiple oligonucleotides. In some examples, the multimer linker 1630 may include between about 3 and about 64 branches, such as the four branches 1633-1 , 1633-2, 1633-3, 1633-4 illustrated by the multimer linker 1630 of FIG. 16F. For example, multimer linkers may include 3 branches, 4, branches, 8 eight branches, 10 branches, 12 branches, 16 branches, 20 branches, or 64 branches, among others. Examples are not so limited and multimer linkers may include more than 64 branches, such as for adding additional dye for signal amplification.

[0238] FIG. 16B illustrates an example composition 1642 comprising a protein 1602, an oligonucleotide 1608, and a tet-TCO 1632. As previously described, the protein 1602 and the oligonucleotide 1608 may be linked via the tet-TCO 1632 (e.g., a tet-modified protein and TCO-oligonucleotide with tet and TCO reacting to form a bond). In some examples, different forms of the composition 1642 may be linked to form a multimer.

[0239] FIG. 16C illustrates an example composition 1644 which is a multimer comprising a protein 1602, a first oligonucleotide 1608-1 , a second oligonucleotide 1608-2, and first and second tet-TCOs 1632-1 , 1632-2. The protein 1602 may be bound to each of the first and second tet-TCOs 1632-1 , 1632-2, with each first and second tet-TCOs 1632-1 , 1632-2 being respectivelybound to one of the first and second oligonucleotides 1608-1 , 1608-2. Examples are not limited to a single protein and two oligonucleotides as shown by FIG. 16C. For example, various compositions may include additional tet, tet-TCOs, proteins, and / or additional oligonucleotides, such as more than two oligonucleotides. As another example, the composition 1644 (and any of compositions described herein) may include a plurality of tets, such as incorporating at least two tets into one protein. As previously described, the first oligonucleotide 1608-1 and second oligonucleotide 1608-2 may be the same or different from one another.

[0240] FIG. 16D illustrates an example composition 1645 which is a SAPO multimer. As shown, the composition 1645 comprises a first oligonucleotide 1608-1 , which may include a single stranded (or hybrid single and double stranded) PS linked to a first protein 1602-1 via a tet-TCO 1632-1 . The composition 1645 further includes second and third oligonucleotides 1608-2, 1608-3 which include single stranded CS that are respectively linked to second and third proteins 1602-2, 1602-3 via tet-TCO 1632-2, 1632-3.

[0241] FIG. 16DD illustrates another example composition 1648 which is a SAPO multimer. The composition 1648 of FIG. 16DD includes at least some of the same features and attributes as, and / or includes an implementation of, the composition 1645 of FIG. 16D but with the first oligonucleotide 1608-1 not being linked to a protein via tet-TCO (e.g., does not include a first protein 1602-1 and tet-TCO 1632-1 of FIG. 16D). As such, and in accordance with various examples, the PS may not include a protein, while the CS may include protein(s). Examples are not limited to the number of tet-TCOs, proteins, and / or oligonucleotides shown by FIGs. 16D-16DD. For example, SAPO multimers may include more or less number(s) of proteins, oligonucleotides, and / or tet- TCOs or other linkers, than illustrated.

[0242] FIG. 16E illustrates another composition 1646 which is a multimer comprising first, second, third, and fourth proteins 1602-1 , 1602-2, 1602-3, 1602-4 and first, second, third and fourth oligonucleotides 1608-1 , 1608-2, 1608-3, 1608-4 which are linked via a multimer linker 1630 that comprises four branches. Each of the first, second, third and fourth proteins 1602-1 , 1602-2,1602-3, 1602-4 and the first, second, third and fourth oligonucleotides 1608-1 , 1608-2, 1608-3, 1608-4 are linked via tet-TCO 1632-1 , 1632-2, 1632-3, 1632-4 as previously described. In some examples, each of the proteins 1602-1 , 1602- 2, 1602-3, 1602-4 may be different form one another, however, examples are not so limited and two or more of the proteins may be the same. Similarly, each of the first, second, third and fourth oligonucleotides 1608-1 , 1608-2, 1608-3, 1608-4 may be different from one another or at least two may be the same. Examples are not limited to the number of tet-TCOs or other linkers, proteins, and / or oligonucleotides shown by FIG. 16E, and example compositions may include more or less numbers of proteins, oligonucleotides, and / or tet-TCOs or other linkers than illustrated. In some examples, the first, second, third, and fourth oligonucleotides 1608-1 , 1608-2, 1608-3, 1608-4 may each (or a portion thereof) include hybrid single stranded and doubled stranded oligonucleotides which are linked to the multimer linker 1630 on a first end and to one of the proteins 1602-1 , 1602-2, 1602-3, 1602-4 on a second end via the tet-TCOs 1632-1 , 1632-2, 1632-3, 1632-4. For example, a single stranded portion of the oligonucleotides may anneal with the multimer linker 1630 and a double stranded portion of the oligonucleotides may be linked to a TCO and react with the tet to link to one of the proteins. In some examples, the first, second, third, and fourth oligonucleotides 1608-1 , 1608-2, 1608-3, 1608-4 may each (or a portion thereof) include each include a pair (e.g., first and second) of single stranded oligonucleotides, wherein a first of the pair of single stranded oligonucleotides is linked to a respective one of the proteins 1602-1 , 1602-2, 1602-3, 1602-4 via the tet-TCOs 1632-1 , 1632-2, 1632-3, 1632-4 and a second of the pair of single stranded oligonucleotides anneal to the first of the pair of single stranded oligonucleotides and to the multimer linker 1630 (e.g., a first portion anneals to the first oligonucleotide and a second portion anneals to the multimer linker 1630).

[0243] FIG. 16F illustrates another composition 1647 which is a multimer comprising first, second, and third proteins 1602-1 , 1602-2, 1602-3 and an oligonucleotide 1608 which are linked via a multimer linker 1630 that comprises four branches 1633-1 , 1633-2, 1633-3, 1633-4. As shown, the oligonucleotide1608 is linked to a first branch 1633-1 of the multimer linker 1630 (directly or optionally through a linker), and the first, second, and third proteins 1602-1 , 1602-2, 1602-3 are respectively linked to the second, third, and fourth branches 1633-2, 1633-3, 1633-4 of the multimer linker 1630 via tet-TCO 1632-1 , 1632-2, 1632-3. In some examples, the tet-TCO 1632-1 , 1632-2, 1632-3 may be linked to the second, third, and fourth branches 1633-2, 1633-3, 1633-4 of the multimer linker 1630 via another linker, such as Dibenzocyclooctyne (DBCO) and / or otherwise using click chemistry as previously described. Examples are not limited to the number of tet-TCOs, proteins, and / or oligonucleotides shown by FIG. 16F, and example compositions may include more or less numbers of proteins, oligonucleotides, and / or tet-TCOs than illustrated.

[0244] In some examples, the composition 1647 of FIG. 16F comprising a multimer linker 1630 may be used when the proteins (e.g., 1602-1 , 1602-2, 1602-3) are the same. In some examples, the composition 1645 of FIG. 16D comprising a SAPO multimer may be used when the proteins (1602-1 , 1602-2, 1602-3) are different from one another. However, examples are not so limited.

[0245] FIG. 16G illustrates an example of a plurality of compositions 1672-1 , 1672-2, 1672-3, 1672-4, 1672-5, 1672-6, 1672-7, 1672-8, 1672-9 (herein generally referred to as “the plurality of compositions 1672”, for ease of reference), which may form part of a kit 1670 and / or otherwise are used to perform signal amplification. Each of the plurality of compositions 1672 may include at least some of substantially the same features and attributes as, or include an implementation of, any of the compositions as previously described by FIGs. 1-16F. For example, each of the plurality of compositions 1672 may include an implementation of the composition of FIG. 1. Accordingly, each composition 1672 may include a protein and an oligonucleotide linked via linker, such as tet-TCO, in a predetermined relationship.

[0246] In various examples, each of the plurality of compositions 1672 include a first component configured to bind to an epitope of the target analyte 1679, a linker, and a second component 1674-1 that provides a signal. For example, using the particular composition 1672-1 , the composition 1672-1 includes the first component 1678-1 , the second component 1674-1 , and the linker 1676-1that links the first component 1678-1 to the second component 1674-1 . In some examples, the plurality of compositions 1672 are configured to bind to a plurality of different epitopes of the target analyte 1679, which may be used to provide signal amplification. For example, the target analyte 1679 may include a biomarker, a complex, a particular cancer target or environment, among other targets which have different epitopes. By using the plurality of compositions 1672 which are configured to bind to different epitopes of the target analyte 1679, each of which are associated with the same signal or with signals that are otherwise additive to one another, the signal is amplified, binding may be improved, and / or the target analyte 1679 may be detected with greater accuracy and / or sensitivity.

[0247] In some examples, each of the first components (e.g., 1678-1 , 1678-2, 1678-3, 1678-4, 1678-5, 1678-6, 1678-7, 1678-8, 1678-9) include proteins and the linkers (e.g., 1676-1 , 1676-2) include oligonucleotides. In some such examples, the proteins may be linked to the oligonucleotides via a tet-TCO reaction, as previously described. Further, in such examples, the second components (e.g., 1674-1 , 1674-2) include signal constructs or signal components, such as fluorophores or multifluors.

[0248] In other examples, each of the first components (e.g., 1678-1 , 1678-2, 1678-3, 1678-4, 1678-5, 1678-6, 1678-7, 1678-8, 1678-9) include oligonucleotides and the linkers (e.g., 1676-1 , 1676-2) include tet-TCO, and optionally, a multimer linker. In such examples, the second components (e.g., 1674-1 , 1674-2) include proteins which act as signal constructs or signal components, such as fluorescent proteins.

[0249] In some examples, at least some of the plurality of compositions 1672 have a 1 :1 protein to oligonucleotide relationship, such as illustrated by the particular compositions 1672-1 , 1672-3, 1672-5, 1672-6, 1672-7, and 1672-9.

[0250] In some examples, at least some of the plurality of compositions 1672 are multimers and have a protein to oligonucleotide relationship of p:1 , 1 :o, or p:o, where p and / or o are greater than 1 , such as illustrated by the particular compositions 1672-2, 1672-4, and 1672-8. In some examples, the multimers may include multiple of the same first component, e.g., a protein oroligonucleotide, which are configured to bind to the same epitope of the target analyte 1679, as illustrated by the particular composition 1672-2 having two of the same first components 1678-2, 1678-3. In some example, the multimers may include multiple different first components, e.g., proteins or oligonucleotides, which are configured to bind to different epitopes of the target analyte 1679, as illustrated by the particular composition 1672-4 having two different first components 1678-4, 1678-5. While FIG. 16G illustrates multimers with two or three first components, examples are not so limited and multimers may include greater than three first components.

[0251] An example method of using the kit 1670 may include a method for interrogating a target analyte. The method may include applying the kit 1670 (e.g., a mixture of protein oligonucleotides) to a sample where the proteins may target different epitopes in a 1 :1 or p:o ratio and / or may contain protein multimers that have a ratio of p:o that is equal to or greater than 2:1 . In some examples, the kit 1670 may include an IS or other oligonucleotide that contains a signal component. For example, the oligonucleotide may contain a single or multiple fluorophores. In some examples, the oligonucleotide that contains the signal component is linked to the same or different protein-oligonucleotide conjugates. In some examples, the method may include performing CycIF, however, examples are not so limited.

[0252] FIGs. 17A-17F illustrate an example SAPO multimer 1750 comprising a PS and a CS, with FIGs. 17B-17F showing different PS examples. As shown by FIG. 17A, the SAPO multimer 1750 may comprise a single stranded PS 1752 and a plurality of single stranded CS 1754-1 , 1754-2, 1754-3, 1754-4. Each CS 1754-1 , 1754-2, 1754-3, 1754-4 may anneal with different portions of the PS 1752. In some instances, at least some of the CS 1754-1 , 1754-2, 1754-3, 1754-4 may anneal with portions of another one of the CS 1754-1 , 1754-2, 1754-3, 1754-4, as shown by the third and fourth CS 1754-3, 1754-4, portions of which anneal with one another and other portions anneal with the PS 1752.

[0253] In some examples, at least some of the CS 1754-1 , 1754-2, 1754-3, 1754-4 are linked to a protein 1702-1 , 1702-2, 1702-3, 1702-4 via a tet 1704-1 , 1704-2, 1704-3, 1704-4 and a TCO 1706-1 , 1706-2, 1706-3, 1706-4 in apredetermined relationship, as previously described. In some examples, as illustrated by FIG. 17A, each of the CS 1754-1 , 1754-2, 1754-3, 1754-4 are linked to a protein 1702-1 , 1702-2, 1702-3, 1702-4, which may be the same or different proteins from one another, or combinations thereof (e.g., two identical proteins, with a third and fourth protein that are different from one another and from the identical proteins).

[0254] The SAPO multimer 1750, as illustrated by FIG. 17A, may be a tree-like structure that may be used or allow for: (i) controlling the spacing between the different proteins via the PS and respective CS; (ii) allow for use of the same or different proteins; and (iii) providing for signal amplification, enhanced binding, and / or improved enzymatic or catalytic reaction. For example, the PS and CS may be designed to control the spacing between different proteins by: (a) the length of the PS, with optional spaces or spacers added between the CS(s) on the PS, (b) adjusting the length of a particular CS with respect to another CS, and (c) creating a fixed distance between different proteins by having the respective CS anneal to one another near the PS, such as shown by the third and fourth proteins 1702-3, 1702-4 in FIG. 17A. The use of the same or different proteins, such as for the first and second proteins 1702-1 , 1702-2, may be used to test for homo- and hetero-antibody binding. For example, the effect of homogeneous and dissimilar dimers, trimers or tetramers on binding to analyte targets may be tested.

[0255] As shown by FIG. 17A, in some examples, the SAPO multimer 1750 may further include a spacer 1756-1 , 1756-2. Examples spacers include an oligonucleotide that is either single or double strand, Polyethylene glycol (PEG), or a specific spacer modifier like LK2128, LK 2129, LK2131 ,or LK2113, among others.

[0256] In some examples, the PS 1752 may take different forms and / or may be linked to a protein, as further illustrated by FIGs. 17B-17F. As noted above, in some examples, the PS 1752 may be completely single stranded. In some examples, the PS 1752 may be a hybrid single stranded and doubled stranded oligonucleotide, where portions are single stranded and other portions are double stranded. In various examples, as illustrated by FIGs. 17B-17D, the PS1752-1 , 1752-2, 1752-3 may be linked to a protein or an aptamer 1758-1 , 1758- 2 that may bind to another protein or target analyte and which is linked to the PS 1752-1 , 1752-2, 1752-3 via tet-TCO 1704, 1706. In some examples, as illustrated by FIG. 17B, the PS 1752-1 may be linked to multiple proteins or aptamers 1758-1 , 1758-2. For example, the first protein or aptamer 1758-1 may bind to the second protein or aptamer 1758-2 that is linked to the tet-TCO 1704, 1706. In some examples, the first protein or aptamer 1758-1 may include a target analyte. In some examples, the protein or an aptamer may float in a solution. In some examples, as shown by FIG. 17D, the protein or an aptamer1758-2 is immobilized on a surface 1760 via at least one linker pair 1759-1 ,1759-2. As shown by FIG. 17E, in some examples, the PS 1752-4 is immobilized on a surface 1760 via at least one linker pair 1759-1 , 1759-2, or as shown by FIGs. 17C and 17F, the PS 1752-4 may float in solution. Examples may include additional variations and combinations thereof as described throughout.

[0257] FIG. 18 illustrates an example SAPO multimer 1860. The SAPO multimer 1860 may comprise a primary SAPO structure 1861 , a secondary SAPO structure 1863, and a tertiary SAPO structure 1865.

[0258] The primary SAPO structure 1861 includes a first oligonucleotide 1808- 1 linked to a first protein 1802-1 via a linker, such as the tet-TCO 1832-1 as illustrated. The oligonucleotide 1808-1 in the primary SAPO structure 1861 may act as a PS, a CS, or a combination thereof. As previously described, the oligonucleotide 1808-1 (or any of the oligonucleotides) may include spacers and may be linked to an aptamer or protein. For example, the oligonucleotide 1808- 1 may be linked to a first protein 1802-1 via tet-TCO 1832-1. While the primary SAPO structure 1861 of FIG. 18 is shown as being linked to the first protein 1802-1 via the tet-TCO 1832-1 , examples are not so limited and in some examples, the primary SAPO structure 1861 may not include a protein (as illustrated by the first protein 1802-1 and tet-TCO 1832-1 being in dashed lines), such as previously illustrated by FIG. 16DD.

[0259] The secondary SAPO structure 1863 includes the primary SAPO structure 1861 annealed to another (or multiple) oligonucleotides. For example,the secondary SAPO structure 1863 may include second and third oligonucleotides 1808-2, 1808-3 which are annealed to the first oligonucleotide 1808-1 of the primary SAPO structure 1861. In some examples, the second and third oligonucleotides 1808-2, 1808-3 are respectively linked to a protein (e.g., the second and third proteins 1802-2, 1802-3) via tet-TCO 1832-2, 1832-3. Although the example illustrates a secondary SAPO structure 1863 with two additional oligonucleotides and proteins, examples are not so limited and secondary SAPO structures may include more or less components, including oligonucleotides and / or proteins, than illustrated by FIG. 18.

[0260] The tertiary SAPO structure 1865 may include a secondary SAPO structure which is built onto the primary SAPO structure 1861 or the secondary SAPO structure 1863. For example, the tertiary SAPO structure 1865 may include the same components as the secondary SAPO structure 1863 as previous described, but with different specific oligonucleotides and proteins. Although the tertiary SAPO structure 1865 is shown as being annealed to the oligonucleotide 1808-1 of the primary SAPO structure 1861 , examples are not so limited and the tertiary SAPO structure 1865 may be annealed to another oligonucleotide, such as the second and / or third oligonucleotides 1808-2, 1808- 3. More specifically, the example tertiary SAPO structure 1865 includes a fourth oligonucleotide 1808-4 linked to a fourth protein 1802-4 via a linker, such as the tet-TCO 1832-4 as illustrated. The fourth oligonucleotide 1808-4, in the particular example, is annealed to the first oligonucleotide 1808-1 of the primary SAPO structure 1861. Fifth and sixth oligonucleotides 1808-5, 1808-6 may be annealed to the fourth oligonucleotide 1808-4. In some examples, the fifth and sixth oligonucleotides 1808-5, 1808-6 are respectively linked to a protein (e.g., the fifth and sixth proteins 1802-5, 1802-6) via tet-TCO 1832-5, 1832-6.Although the example illustrates a tertiary SAPO structure 1865 with three oligonucleotides and proteins, examples are not so limited and tertiary SAPO structures may include more or less components, including oligonucleotides and proteins, than illustrated by FIG. 18. Further, the multimer 1860 may include additional tertiary SAPO structures.

[0261] In some examples, the components of primary SAPO structure 1861 , secondary SAPO structure 1863, and / or the tertiary SAPO structure 1865 may form part of a kit. In some examples, the secondary SAPO structure 1863 may act as a CS or a combination of a CS to the PS and a PS for another CS (e.g., secondary oligonucleotides, such as another tertiary SAPO structure). The kit may contain the first oligonucleotide 1808-1 (e.g., the PS), second and third oligonucleotides 1808-2, 1808-3 that are complementary to a portion of the first oligonucleotide 1808-1 (and may be a PS to another second oligonucleotides) which may be used for spacing, and / or the oligonucleotides 1808-4, 1808-5, 1808-6 of the tertiary SAPO structure 1865. The PS may serve as the base for the CS(s) to anneal to. In some examples, the kit may further include the different proteins and linkers, e.g., tet-TCO.

[0262] Various examples are directed to methods of forming any of the abovedescribed compositions. An example method for forming a composition comprising a protein-oligonucleotide conjugate includes generating a tet- modified protein which includes a protein that incorporates a tet. For example, the protein may be generated as described by WO 2016 / 176689 (PCT / US2016 / 030469), including but not limited to expressing, purifying, and characterizing the protein that includes a tet-amino acid. The method further includes obtaining an oligonucleotide with a set length and sequence, and having a functional group on a terminal end. The oligonucleotide may be formed using conventional techniques and / or purchased. The method may optionally include converting the functional group on the terminal end of the oligonucleotide to TCO. And, the method includes conjugating the tet-modified protein to the oligonucleotide-TCO.

[0263] The above may be used to form a protein-oligonucleotide conjugate with a ratio of protein to oligonucleotide of 1 :1. In some examples, a protein- oligonucleotide conjugate may be formed which is a multimer. In such examples, the above method further includes forming a plurality of conjugates, such as CS(s), as described above. In some examples, the method includes generating a PS which includes sequences complementary to the CS(s) and annealing each of the CSs to the PS.

[0264] In some examples, the above described method may be used to make a multimer with a multimer linker. For example, the method may further include obtaining a multimer linker with a set number of branches, length of branches, and optionally, with differentiated functional groups on the branches (e.g., at least two sets). The method may further include converting at least one set of functional groups on the multimer linker to TCO or to otherwise converted to react with tet. In some examples, optionally the method includes converting another set of the functional groups to be configured to react with the oligonucleotide (e.g., to directly react and link). Similarly to that described above, the method may include conjugating the tet-modified protein(s) to the multimer linker via the TCO on the branch(s) and conjugating the oligonucleotide to the oligo-functional branch of the multimer linker either simultaneously or sequentially.

[0265] In any of the example methods, the proteins may include full proteins or protein fragments, such as sdAb, scFv, and Fabs. Additionally, dyes or other signal components may form part of the compositions, such as an oligonucleotide or protein with multiple dyes. Further, the above methods may involve different types of reactions, such as SPAAC, IEDDA, acid / base reacts, and / or annealing of oligonucleotides. In some examples, the conjugation reaction may include the reaction type of IEDDA, which may be both efficient in terms of reagent use and time for the reaction to occur. For example, the conjugation rection may be completed over a time period of between about 0.5 hours and about 8 hours, and in some instances, about 0.5 hours and about 5 hours, about 0.5 hours and 2 hours, or about 0.5 hours and 1 hour and such that the final assembled composition may be formed in less than twenty four hours, such as between about 12 hours and about twenty four hours. In comparison, prior techniques often day multiple days. Similarly, such reactions may not require excess reagents, thereby saving on both time and costs.

[0266] In any of the above described examples, the compositions formed may reduce the energy, time, and / or costs required to develop viable assays. Furthermore, the time required to evaluate an assay may be reduced with a known volume and the resulting population (or volume) of protein-oligonucleotide conjugates may include a single species due the predetermined relationship of protein to tet. The single species may increase the quality of the protein-oligonucleotide conjugates formed due to the quantitative loading of the oligonucleotide. Furthermore, in some examples, direct labeling of the conjugate may avoid errors in the binding process due to the ability to maintain protein stability. In various examples, the single species of reagent, e.g., the protein- oligonucleotide conjugate, may have the same binding characteristics and may be formed in a fraction of time as compared to prior techniques. In some examples, the resulting conjugates may have greater purity, yield, and / or ease of forming compared to prior techniques. With imaging reagent-applications, the DS / IS or other types of PS / CS pairs may be closer to the target analyte, which may allow for more precise and / or sharper imaging. For example, the oligonucleotide starts at the same spot and the resulting fluorophore may be as a consistent distance across the species. For changing charge and / or mass reagent-applications, the ease of design for multiple charges and / or masses due to the predetermined relationship for binding and as the orientation is consistent. In any reagent-application type, there may be less variability from run to run, with reduced variability in the detectable signal such that postprocessing may have higher resolution and / or lower variance. In some examples, the resulting reagents for a reagent-application may be more deterministic and / or predictable, and allow for a defined range of molecular weights of proteins, selectable length of the oligonucleotide, and 1 :1 , 1 :n, p:1 , or p:n protein to tet coupling.

[0267] The following examples may comprise at least some of substantially the same features and attributes as, and / or example implementations of, the previously described examples of the present disclosure. The following examples may be implemented alone or together, which may comprise any various complementary combinations.

[0268] Example A1 : A non-transitory computer-readable medium comprising instructions that are executable to cause a processing circuitry to: provide a user interface including a display of a plurality of types of reagent-applications; in response to user input selecting a reagent-application of the plurality of typesof reagent-applications, revise the user interface to display a plurality of subtypes of the selected type of reagent-application; and in response to user input indicating a sub-type of the plurality of sub-types, further revise the user interface to display a plurality of potential proteins or aptamers associated with the selected sub-type.

[0269] Example A2: The non-transitory computer-readable medium of Example A1 wherein the plurality of sub-types include different targets for the selected type of reagent-application.

[0270] Example A3: The non-transitory computer-readable medium ofExample A1 , wherein the display of the plurality of sub-types includes selectable icons, dropdown boxes, and / or an input field.

[0271] Example A4: The non-transitory computer-readable medium of Example A1 , wherein the display of the plurality of potential proteins or aptamers associated with the selected sub-type are automatically populated by the processing circuitry using data indicative of available proteins or aptamers configured to bind to a target analyte associated with the selected sub-type of the selected type of reagent-application.

[0272] Example A5: The non-transitory computer-readable medium of Example A1 , wherein the display of the plurality of potential proteins or aptamers further includes an input field for a user to input a protein or aptamer not included in the plurality of potential proteins or aptamers which are automatically populated.

[0273] Example A6: The non-transitory computer-readable medium of Example A1 , wherein the display of the plurality of potential proteins or aptamers includes an input field for a user to input a protein or aptamer.

[0274] Example A7: The non-transitory computer-readable medium of Example A5 or Example A6 further including instructions executable to, responsive to user input indicating the protein or aptamer, store data indicating the protein or aptamer is associated with the sub-type of the selected type of reagent-application.

[0275] Example A8: The non-transitory computer-readable medium of Example A1 , further including instructions executable to, responsive to userinput selecting a set of proteins or aptamers from the plurality of potential proteins or aptamers and / or as manually input, revise the user interface to display the selected set of proteins or aptamers with oligonucleotides.

[0276] Example B1 : A non-transitory computer-readable medium comprising instructions that are executable to cause a processing circuitry to: set a number of cycles for performing a reagent-application based on received user input indicative of: a number of proteins for the reagent-application; and a number of signals for the reagent-application, wherein the number of cycles is dependent on the number of signals and the number of proteins; provide a user interface including a display of the set number of cycles with the number of signals for each of the number of cycles; and in response to user input, assign the number of proteins to one of the number of cycles and one of the number of signals.

[0277] Example B2: The non-transitory computer-readable medium of Example B1 , further including instructions executable to revise the user interface to display the assignment in response to the user input.

[0278] Example B3: The non-transitory computer-readable medium of Example B1 , further including instructions executable to: identify a plurality of potential proteins responsive to received user input indicative of a type of reagent-application; and display the plurality of potential proteins for selecting by a user via the user interface.

[0279] Example C1 : A non-transitory computer-readable medium comprising instructions that are executable to cause a processing circuitry to: provide a user interface including a display of a plurality of potential proteins associated with a mobility modulation reagent-application and a target; in response to user input to select a number of the plurality of potential proteins, revise the user interface to display indications for user input to select: a length of oligonucleotides to be linked to the selected number of the plurality of potential proteins; and the oligonucleotides being single stranded, double stranded, or a combination thereof; and in response to user input to select the length of the oligonucleotides and the oligonucleotides being single stranded and / or double stranded, assign the selected number of the plurality of potential proteins to oligonucleotides and signal constructs.

[0280] Example C2: The non-transitory computer-readable medium of Example C1 , wherein the instructions to revise the user interface to display indications for user input to select further including instructions executable to revise the display to provide user input to select: the oligonucleotides including deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA); a length of second oligonucleotides which are respectively complementary to one of the oligonucleotides; wavelength, fluorophore emission, and / or fluorophore excitation; the second oligonucleotides; and / or mass or charge change associated with complexes formed by proteins linked to oligonucleotides which bind to target analytes associated with the target.

[0281] Example C3: The non-transitory computer-readable medium of Example C1 , wherein the revision to the user interface includes a set of sequential displays to provide user input for each selection, selection in one display causing the next display to be provided.

[0282] Example C4: The non-transitory computer-readable medium of Example C1 , further including instructions executable to revise the user interface to display the assignment in response to the user input.

[0283] Example D1 : A non-transitory computer-readable medium comprising instructions that are executable to cause a processing circuitry to: provide a user interface including a display of a plurality of potential proteins associated an imaging reagent-application of a target; and in response to user input to select a number of the plurality of potential proteins, revise the user interface to display indications for user input to select: a wavelength; fluorophore emission; fluorophore excitation; a length of oligonucleotides to be linked to the selected number of the plurality of potential proteins; and / or a length of second oligonucleotides which are respectively complementary to one of the oligonucleotides; and in response to user input to select the wavelength, fluorophore emission, fluorophore excitation, length of the oligonucleotides, and / or length of the second oligonucleotides, assign the selected number of proteins to an oligonucleotide of the selected oligonucleotides, optionally a second oligonucleotide of the selected second oligonucleotides, and a signalconstruct associated with the selected wavelength, fluorophore emission, and fluorophore excitation.

[0284] Example D2: The non-transitory computer-readable medium of Example D1 , further including instructions executable to revise the user interface to display the assignment in response to the user input.

[0285] Example E1 : A method comprising applying a mixture of protein and oligonucleotides (e.g., a kit) to a sample, wherein the proteins target different epitopes in a 1 :1 or p:o ratio and / or contain protein multimers that have a ratio of p:o of equal to or greater than 2: 1 .

[0286] Example E2: The method of Example E1 , wherein the mixture of protein and oligonucleotides include an IS or other oligonucleotide that contains a signal component.

[0287] Example E3: The method of claim E2, wherein the oligonucleotide contains a single or multiple fluorophores.

[0288] Example E4: The method of Example E2, wherein oligonucleotide that contains the signal component is linked to the same or different protein- oligonucleotide conjugates.

[0289] Example E5: The method of Example E1 , wherein the method includes performing CycIF.

[0290] Example F1 : A method of screening for multiple target analytes in a single test using a biological sample, the method including combining multiple protein-oligonucleotide conjugate with the biological sample and running the sample on at least one lanes of an electrophoresis device.

[0291] Example F2: The method of Example F1 , further include concentrating the biological sample

[0292] Example F3: The method of Example F1 , wherein each protein- oligonucleotide conjugate bound to one of the multiple target analytes in the biological samples has a unique mass, charge, and / or charge and mass.

[0293] Example G1 : A method for ordering protein-oligonucleotide conjugates or other compositions including providing a user interface including a display with of the plurality of types of reagent-applications for the user to select; in response to the user selecting one of the reagent-applications in the userinterface, revising the user interface to display with the options to selected a pre-tested kit, an experimental tool kit, or a combination for purchasing; in response to the user selecting one of the options, and if an experimental tool kit or a combination is selecting, in response to prompting the user through options, providing an output to the manufacturer indicating the type of reagentapplication, the type of kit, and the components for the kit for generating the kit for the user.

[0294] Example G2: The method of Example G1 , further including prompting the user to select how the protein-oligonucleotide conjugates or other compositions are delivered.

[0295] Example G3: The method of Example G1 , further including prompting the user to select how many oligonucleotides to connect together and spacing requirements.

[0296] As used herein, designations of “first”, “second”, “third”,” are used to refer to one element and another of the same element, of the same type or of a different type, without reference to temporal order.

[0297] For more general information on proteins attached to tet and specific information on techniques to immobilize tetrazine on porous membranes, reference is made to: US Patent Publication 2021 / 0072238, published on March 11 , 2021 , and entitled “Immobilization of proteins with controlled orientation and load”, which is herein incorporated by reference in its entirety for its teachings; and to WO 2022 / 109075 (PCT / US2021 / 059798), published on May 27, 2022, and entitled “Configurable Substrate of a Fluidic Device”, which is herein incorporated by reference in its entirety for its teachings. For more specific and general information on the preparation of representative tetrazine non-canonical amino acids, methods for genetic encoding proteins and polypeptides using the tetrazine non-canonical amino acids, and proteins and polypeptides comprising the tetrazine non-canonical amino acids reference is made to WO 2016 / 176689 (PCT / US2016 / 030469), published on November s, 2016, and entitled “Reagents and methods for bioorthogonal labeling of biomolecules in living cells”, which is herein incorporated by reference in its entirety for its teachings.

[0298] For purposes of this application, “processor” or “processing circuitry” shall mean a presently developed or future developed processor (or processing resources) that executes computer-readable instructions contained in a memory or that includes circuitry to perform computations. In some examples, execution of the computer readable instructions, such as those provided via memory, cause the processing resource to perform the above-identified actions, such as providing a user interface in the various example implementations as generally described in (or consistent with) at least some examples of the present disclosure. The computer-readable instructions may be loaded in a RAM for execution by the processor from their stored location in a ROM, a mass storage device, or some other persistent storage (e.g., non-transitory tangible medium or non-volatile tangible medium), as represented by memory. The computer- readable instructions may include a sequence of instructions, a processorexecutable machine learning model, or the like. In some examples, memory comprises a computer-readable tangible medium providing non-volatile storage of the computer-readable instructions executable by processing circuitry. In some examples, the resources (e.g., processing circuitry and / or memory) may be distributed, such as with cloud computing or other wireless applications. In some examples, the computer-readable tangible medium may sometimes be referred to as, and / or comprise at least a portion of, a computer program product. In other examples, hard wired circuitry may be used in place of or in combination with machine readable instructions to implement the functions described. For example, processing circuitry and / or memory may be embodied as part of at least one application-specific integrated circuit (ASIC), at least one field-programmable gate array (FPGA), and / or the like. In at least some examples, the processing circuitry and / or is not limited to any specific combination of hardware circuitry and computer-readable instructions, nor limited to any particular source for the computer-readable instructions executed by the processing circuitry.

[0299] The skilled artisan would recognize that various terminology as used in the Specification (including claims) connote a plain meaning in the art unless otherwise indicated. As examples, the Specification describes and / or illustratesaspects useful for implementing the claimed disclosure by way of various circuits or circuitry which may be illustrated as or using terms such as blocks, modules, device, system, unit, processing circuitry, memory and / or other circuittype depictions (e.g., reference numerals 1050 and 1060 of FIG. 10A depict a block / module as described herein). Such circuits or circuitry are used together with other elements to exemplify how certain examples may be carried out in the form or structures, steps, functions, operations, activities, etc. In certain examples, such a programmable circuit is one or more computer circuits, including memory circuitry for storing and accessing a program to be executed as a set (or sets) of instructions (and / or to be used as configuration data to define how the programmable circuit is to perform), and an algorithm or process as described at FIG. 10 is used by the programmable circuit to perform the related steps, functions, operations, activities, etc. Depending on the application, the instructions (and / or configuration data) may be configured for implementation in logic circuitry, with the instructions (whether characterized in the form of object code, firmware, or software) stored in and accessible from a memory (circuit).

[0300] The various ranges provided herein include the stated range and any value or sub-range within the stated range. Furthermore, when “about” is utilized to describe a value, this includes, refers to, and / or encompasses variations (up to + / — 10%) from the stated value.

[0301] Although specific examples have been illustrated and described herein, a variety of alternate and / or equivalent implementations may be substituted for the specific examples shown and described without departing from the scope of the present disclosure. This application is intended to cover any adaptations or variations of the specific examples discussed herein. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof.Experimental Embodiments

[0302] As further illustrated below in connection with experimental examples, different protein-oligonucleotide compositions were formed and / or used to assess use for different reagent-applications.

[0303] In various experiments, protein-oligonucleotide conjugates were formed that included sdAbs that incorporated tet and which were linked to an oligonucleotide via TCO. The resulting conjugates exhibited a predetermined relations of protein to tet of 1 :1 and resulted in a ratio of protein to oligonucleotide of 1 :1. The protein-tet reacted with the TCO-oligonucleotide at room temperature in the dark for two hours. Table 1 below shows the reactants, concentrations, and amounts used in the reaction.Table 1

[0304] The protein-oligonucleotide conjugates formed were used in an imaging reagent-application and without purification in various experiments. In particular, sdAbl and sdAb2 were sdAb designed to bind to a spike protein expressed in cells. sdAb3 was not designed to bind to the spike protein.

[0305] FIGs. 19A-21 are images from an example imaging reagent-application implementation, which was applied using the protein-oligonucleotide conjugates from Table 1. In particular, for each sdAb, sdAb-DS was prepared and annealed to-IS with fluorophore, indicated as sdAb-DS- S with fluorophore. In the example experiments, negative controls were assessed using the sdAb2-tet and the sdAb3-tet. Negative controls were assessed by using the sdAb2-DS-IS with fluorophore AZ594 to assess a sample that does not contain the spike expressed in cells and using sdAb3-DS-IS with fluorophore AZ488 on a sample containing the spike expressed in cells.

[0306] FIGs. 19A-19B illustrates the results of the negative controls. For example, FIG. 19A is a fluorescent image of a sample that does not contain the spike expressed after exposing the sample to the sdAb2-DS-IS with fluorophore AZ594. As noted above, sdAb2 is designed to bind to the spike. As the spike does not exist in the sample, it would be expected that sdAb2-DS-IS with fluorophore AZ594 would not stain, which is shown by FIG. 19A not having any red signal. As may be appreciated, the blue signal may show staining of the nucleus of the cells by DAPI (blue channel). As the sdAb2-DS-IS with fluorophore AZ594 did not stain, this indicates that there is minimal or no nonspecific binding with the set of reagents.

[0307] FIG. 19B is a fluorescent image of a sample that contains the spike expressed in cells after exposing the sample to the sdAb3-DS-IS with fluorophore AZ488. As previously described, sdAb3 is not specific for the spike, and it would be expected that sdAb3-DS-IS with fluorophore AZ488 would not stain a sample that contains the spike. If there was staining, this would indicate nonspecific binding. As shown by FIG. 19B, the sdAb3-DS-IS with fluorophore AZ488 did not stain the sample, indicating the sdAb3-DS-IS with fluorophore AZ488 has minimal or no nonspecific binding.

[0308] In some experiments, sdAb1-DS-IS with fluorophore AZ488 and sdAb2- DS-IS with fluorophore AZ488 were exposed to samples containing cells that express the spike. FIG. 20A shows the resulting fluorescent image of the sample containing the spike after exposing the sample to sdAb1-DS-IS with fluorophore AZ488 and FIG. 20B shows the resulting fluorescent image from the sample containing the spike after exposing the sample to sdAb2-DS-IS with fluorophore AZ488. Both images in FIGs. 20A-20B show staining with AZ488 (e.g., the green channel), successfully showing the protein-oligonucleotide conjugates and IS being used to image a target analytes.

[0309] In some experiments, a sample that expressed the spike was exposed to sdAb1-DS-IS with fluorophore AZ488, sdAb2-DS-IS with fluorophore AZ594, and DAPI, and images in green, red, and blue channels, respectively. FIG. 21 shows the resulting fluorescent image after from the exposing the sample, whichillustrates the combined signals from AZ488 and AZ594 (e.g., yellow signal) and the blue signal.

[0310] Some experiments were directed to using protein-oligonucleotide conjugates in a mobility modulation reagent-application. Different protein-PS were generated using green fluorescent protein (GFP) and single stranded DNA PS of different lengths. For example, the following protein and protein-PS were input to a gel and electrophoretic separation was performed: GFP was conjugated with TCO without nucleotides (control), GFP-PS of 5 nucleotides, GFP-PS of 10 nucleotides, and GFP-PS of 20 nucleotides.

[0311] FIG. 22 is an image of a gel from a mobility modulation reagentapplication demonstration experiment with the above protein and protein-PS. In the image, lanes 3-4 are GFP (control), lanes 5 and 8 are GFP-PS of 5 nucleotides, lanes 6 and 9 are GFP-PS of 10 nucleotides, and lanes 7 and 10 are GFP-PS of 20 nucleotides.

[0312] As shown in the image of FIG. 22, the GFP control was separated to 27kDa and migrated. As further shown, the addition of different length PS caused the band shift relative to the GFP control on lanes 3-4, with longer PS (e.g., lanes 7 and 10) leading to greater band shift than shorter PS (e.g., lanes 5 and 8). Additionally shown are bands from waste or other undesired products, which are believed to result from impurities in the DNA-TCO starting material reacting with GFP-tet.

[0313] Various experiments were conducted to show tet incorporation by a protein. In such experiments, a tet-amino acid was successfully incorporated into a variety of proteins, at specified location(s), and with one tet or two tets. Such proteins included a fluorescent protein (GFP), an sdAb (NB22), an enzyme (lactate oxidase), and a protein (Protein A). Additionally experiments were conducted with scFv and Fab.

[0314] FIGs. 23A-23B and 24 are images of gels from tet incorporation experiments. As shown, tet was incorporated into GFP and NB22 and then electrophoretic separation was performed. FIG. 23A is an image of a gel that demonstrates incorporation of tet by GFP. FIG. 23B is an image of a gel that demonstrates incorporation of tet by NB22. Alternatively and / or in addition toperforming electrophoretic separation, mass spectrometry may be used to determine the incorporation of the tet into a protein.

[0315] FIG. 24 is an image of a gel that demonstrates incorporation of tet by an enzyme of lactate oxidase. As shown, the experiments included performing electrophoretic separation on a wild type lactate oxidase, and tet incorporated by lactate oxidase at three different locations, respectively at amino acid positions 188, 207, and 254. The shifting of the bands upon conjugation with TCO-PEG5K-TCO showed doubling of molecular weight of lactase oxidase and indicated successful incorporation of tet at the three different locations. The different lanes of the gel show the wild type (wt) lactate oxidase, incorporated tet at position 188 of lactate oxidase, incorporated tet and TCO-PEG5K-TCO at position 188 of lactate oxidase, incorporated tet at position 207 of lactate oxidase, incorporated tet and TCO-PEG5K-TCO at position 207 of lactate oxidase, incorporated tet at position 254 of lactate oxidase, and incorporated tet and TCO-PEG5K-TCO at position 254 of lactate oxidase.

[0316] FIG. 25 is an image of a gel from a tet incorporation experiment with protein A and one tet or two tets. As shown, protein A was modified to incorporate a tet at A43 or a first tet at A43 and a second tet at K58, and then electrophoretic separation was performed. The different lanes of the image show the ladder, protein A with tet at A43, protein A with tet and TCO-PEG5K- TCO at A43, protein A with a first tet at A43 and a second tet at K58, protein A with first and second tets and TCO-PEG5K-TCO at A43 and at K58, and background. The gel showed protein A incorporated the tet(s). In particular, the one tet incorporation showed a shift from protein A after conjugated with TCO- PEG5K-TCO, demonstrating successful incorporation of the tet moiety. The two tet incorporation showed a doubled shift as compared to incorporating one tet and one sTCO-PEG.

[0317] Various experiments were conducted to show the generation of protein- oligonucleotide conjugates (PrOCs) from a variety of proteins and repeatability of generating the same. The repeatability was good as measured by the consistency of the degree of conjugation (DOC). Such experiments showed the successful generation of several PrOCs from different proteins and theconsistency of DOC. Although the various experiments show the PrOCs formation through mobility via electrophoretic separation, mass spectrometry may alternatively and / or additionally be used.

[0318] FIGs. 26A-26C are images of gels showing PrOCs with one protein and one oligonucleotide. Each of FIGs. 26A-26C are images of Coomassie gels illustrating the mobility of an unconjugated protein-tet and a protein-tet- oligonucleotide conjugate with different oligonucleotide base lengths and responsive to performing electrophoretic separation. In the images of FIGs. 26A-26B, lane 4 is no oligonucleotide, lane 5 is an oligonucleotide with 10 bases, lane 6 is an oligonucleotide with 20 bases, lane 7 is an oligonucleotide with 28 bases, lane 8 is an oligonucleotide with 34 bases, and lane 9 is an oligonucleotide with 96 bases. In FIG. 26C, lane 2 is no oligonucleotide, lane 3 is an oligonucleotide with 5 bases, lane 4 is an oligonucleotide with 10 bases, lane 5 is an oligonucleotide with 20 bases, lane 6 is an oligonucleotide with 28 bases, and lane 7 is an oligonucleotide with 34 bases. More specifically, FIG. 26A is an image of a gel showing unconjugated dGFP-tet and a PrOC with dGFP-tet in response to performing electrophoretic separation. FIG. 26B is an image of a gel showing unconjugated aA1 scFv-tet and a PrOC with aA1 scFv- tet in response to performing electrophoretic separation. FIG. 26C is an image of a gel showing unconjugated aPD-L1 VHH6 sdAb-tet and a PrOC with aPD-L1 VHH6 sdAb-tet in response to performing electrophoretic separation. The PrOCs used in the experiment illustrated by the images of FIGs. 26A-26C included PrOC A constructs, as further described below.

[0319] To evaluate the consistency of DOC of the PrOC, two tet-containing proteins, one scFv and one sdAb, were conjugated to a TCO-AZ594. Excess dye was removed and the absorbance of the samples at 280 nanometers (nm) and 592 nm, corresponding to the peak absorbance of the protein and dye, were measured three times each using a Nanodrop spectrophotometer. A calibrated correction factor was used to subtract the contribution of the dye to absorbance at 280nm. The resulting DOC is the molar ratio of the dye over the protein. This process was repeated for another independent conjugation lot of each protein. The mean values and percent (%) coefficient of variation (CV)were calculated for each lot. As shown by Table 2 below, the mean DOC between the two lots of conjugation were similar and the % CV were around or below 10%, considered good for variability of general samples. The overall (all data considered of the two lots) % CV is good for scFv and fairly good for VHH6.Table 2

[0320] The demonstrated mobility relationship between the length of the oligonucleotide of a PrOC and the distance travelled on a gel may be used to separate and detect a mixture of biological targets using electrophoresis. FIG. 26A-26C show the change in mobility based on the additional length of the oligo added to a single protein. Electrophoresis tests may include affinity electrophoresis, capillary electrophoresis, electroblotting, electrophoretic mobility shift assay, free-flow electrophoresis, native PAGE, SDS- Page, pulsed- field gel electrophoresis, IEF (IsoElectric Focusing) electrophoresis, two- dimensional Gel Electrophoresis and column electrophoresis, among others. For a given mixture of biological targets, a set of PrOC may be prepared with each binding to a specific target with a unique oligonucleotide length. When tested in the electrophoretic system, these biological targets / PrOCs separate based on the mass of the Target + PrOC and the Target + PrOC charge. The sample solution may or may not need to be concentrated. For example, one simple urine test may detect kidney cancer, prostate cancer and / or bladder cancer. If a urological cancer was suspected, the anti-VHL-PrOC, anti-PSA- PrOC or anti-PSAM-PrOC, and the anti-NMP22-PrOC may be combined withthe sample and an electrophoresis test may be run on the sample and PrOC mixture. The PrOCs within the mixture bind to their respective targets (if available). The separation of these targets in a single lane or multiple lanes from the same sample can easily determine which cancer was present. Kits can be made that are ready to test urine samples of patients suspected of having urological cancer. The advantage of using a kit is that these PrOCs have been designed to work together for specific applications, meaning that the proteins or protein fragments do not interact with each other, and only react with one CS.

[0321] FIGs. 27A-27C are images of gels showing mobility of the PrOCs with one protein and one oligonucleotide, as described above in connection with FIGs. 26A-26C, but as stained with SYBR gold to show the oligonucleotide. In particular, FIG. 27A is an image of gel showing unconjugated dGFP-tet and a PrOC with dGFP-tet after performing electrophoretic separation. FIG. 27B is an image of gel showing unconjugated aA1 scFv-tet and a PrOC with aA1 scFv-tet after performing electrophoretic separation. FIG. 27C is an image of gel showing unconjugated aPD-L1 VHH6 sdAb-tet and a PrOC with aPD-L1 VHH6 sdAb-tet after performing electrophoretic separation. The PrOCs used in the experiment illustrated by the images of FIGs. 27A-27C included PrOC A constructs, as further described below.

[0322] As shown by the images of the gels in FIGs. 26A-27C, several proteins with variable molecular weights (15kDa to 28kDa, for example) and theoretical charges (nearly zero to -7 in the buffer solutions used) were conjugated with oligonucleotides of a number of lengths (5, 10, 20, 28, 34 and 96) and their mobilities evaluated on TGX gel stained with SYBR gold (to visualize oligo) and Coomassie (to visualize protein) respectively. As supported by the images, increasing oligonucleotide length led to increased mobility (e.g., moved further down the gel). Surprisingly, this behavior reached a plateau around 28 nucleotides (nt) for most of the proteins tested and eventually reversed. For GFP, the most negatively charged and the larger one of the proteins tested, the plateau was reached at 20 nt. This plateauing behavior held for several repeated conjugation lots. In some examples, this plateau may be used as a threshold for providing distinguishing mobility for different conjugates or formedcomplexes. For example, this threshold may be used within a user interface, as discussed above.

[0323] FIGs. 28A-28B are images of gels showing mobility of the PrOCs as previously described by FIGs. 26A and 27A. That is, the gels show mobility of unconjugated dGFP-tet and a PrOC with dGFP-tet, with mobilities evaluated on TGX gel stained with SYBR gold (FIG. 28A) and Coomassie (FIG. 28B). The gels show repeatability of PrOC with dGFP-tet, as previously shown by FIGs. 26A and 27A. The PrOCs used in the experiment illustrated by the images of FIGs. 28A-28B included PrOC A constructs, as further described below. Table 3 summarizes the lanes from the gels.Table 3

[0324] Various experiments were directed to generating PrOCs at different p:o ratios using either a multimer linker or through SAPO. With SAPO, experiments were conducted to show annealing an IS to a SAPO protein dimer on a PS.

[0325] FIG. 29 is an image of a gel showing mobility of a multimer comprising a PrOC with one protein and three oligonucleotides linked via a multimer linker. In the image, lanes 1 and 2 were used as controls, with lane 1 including the protein ladder and lane 2 including scFv-tet. Lanes 3 and 12 were empty. Lanes 4-7 included scFv-tet + TCO-PEG-3XAzide at different ratios: lane 4 was 1 :1 / 4, lane 5 was 1 :1 / 2, lane 6 was 1 :1 , and lane 7 was 1 :2. Lanes 8-11 includes oligonucleotide-DBCO + TCO-PEG-3XAzide + scFv-tet at different ratios: lane 8 was 1 : 1 / 6: 1 , lane 9 was 1 : 1 / 3: 1 , lane 10 was 1 :1 :1 , and lane 11 was 1 :2: 1 . A band around 35K was the desired product. This gel showed that one protein (e.g., anti-hHbA1 scFv) to three oligonucleotides can be linked together using a multimer linker. The PrOCs used in the experiment illustrated by the image of FIG. 29 included PrOC B constructs, as further described below.

[0326] FIGs. 30A-30B are images of gels showing mobility of a multimer comprising a SAPO with three PrOCs and a PS. In particular, the three PrOCs included three oligonucleotide-protein conjugates which annealed to a PS to self-assemble. The PrOCs used in the experiment illustrated by the images of FIGs. 30A-30B included PrOC D constructs, as further described below. Tables 4 and 5 summarize the lanes of FIGs. 30A-30B.Table 4Table 5

[0327] FIGs. 31 A-31 B are images of gels showing mobility of a multimer comprising a SAPO with two PrOCs, one IS, and a PS. In particular, the two PrOCs included two oligonucleotide-protein conjugates and the IS included an oligonucleotide-dye which each annealed to the PS to self-assemble. As such, a SAPO dimer can be imaged by using the IS. Table 6 summarizes the lanes of FIGs. 31 A-31 B, with FIG. 31 A including an unstained gel and FIG. 31 B including a gel stained with SYBR gold. The CS-dGFP or CS-594 containing product and unreacted CS-Azide could be removed by clean-up steps. The PrOCs used in the experiment illustrated by the images of FIGs. 31 A-31 B included PrOC E constructs, as further described below.Table 6

[0328] Various experiments were conducted to assess for application of the compositions described herein for signal amplification. In particular, multifluors were generated for signal amplification via the use of a multimer linker and / or SAPO. The term “multifluor” refers to a multimer or complex formed that has multiple fluorophores.

[0329] FIGs. 32A-32B are images of gels showing mobility of a multifluor formed using a multimer linker. In particular, the multifluor included a multimer linker having four branches that linked one protein to three dye. According, the multifluor had three dyes. The compositions used in the experiment illustrated by the images of FIGs. 32A-32B included F constructs, as further described below. Additional compositions were generated that includes one oligonucleotide and three dye referred to as G constructs, as further described below.

[0330] FIG. 32A was generated by performing electrophoretic separation including 8-16% reducing TGX gel, 200 Volts (V), 30 minutes, UV light. As shown by FIG. 32A, lane 1 was the ladder, lane 2 was sfGFP-N150 (not visible), lane 3 was sfGFP-N150 + TCO-amine, lane 4 was the multifluor (e.g., CF488A product). Lanes 5-9 included sfGFP-N150 + TCO-PEG-3xCF488A at different ratios, including 1 :1 / 6 in lane 5, 1 : 1 / 3 in lane 6, 1 :1 in lane 7, and 1 :2 in lane 8. Lanes 9-12 included sfGFP-N150+ TCO-PEG-3xCF594 at different ratios, including 1 :1 / 6 in lane 9, 1 : 1 / 3 in lane 10, 1 :1 in lane 11 , and 1 :2 in lane 12.

[0331] FIG. 32B was generated by performing electrophoretic separation including 8-16% native TGX gel, Coomassie stained. As shown by FIG. 32B, lane 1 and lane 2 were controls, including dGFP in lane 1 , and dGFP + TCO- amine in lane 2. Lanes 4-6 included multifluors with CF594, including dGFP + TCO-PEG-3xCF594 (1 :0.25) in lane 4, dGFP + TCO-PEG-3xCF594 (1 :0.5) in lane 5, and dGFP + TCO-PEG-3xCF594 (1 :1 ) in lane 6. Lanes 8-10 included multifluors with CF488A, including dGFP + TCO-PEG-3xCF488A (1 :0.25) inlane 8, dGFP + TCO-PEG-3xCF488A (1 :0.5) in lane 9, and dGFP + TCO-PEG- 3xCF488A (1:1) in lane 10.

[0332] FIGs. 33A-33B are images of gels showing mobility of multifluors comprising a SAPO. In particular, the SAPO included three IS annealed to a PS FIGs. 33A-33B demonstrated annealing multiple CS that have been conjugated with multiple dyes to a PS resulting in a SAPO multifluor which may be used to amplify signals thorough the linkage of the PS to the target. The SAPO used in the experiment illustrated by the images of FIGs. 33A-33B included SAPO C constructs, as further described below. Table 7 summarizes the lanes:Table 7

[0333] Various experiments were conducted for purification. For example, a variety of cleanup methods can be employed to remove unreacted TCO-DBCO, azide-oligo, and TCO-oligo, for example, Oligo Clean & Concentrator kit from Zymo, reversed-phase cartridge purification, HPLC oligo purification, Vivaspin desalting columns or size exclusion columns like Zeba Spin desalting columns.

[0334] The above described PrOC and other compositions were generated as follows. For PrOCs having a 1 :1 ratio of protein to oligonucleotide a tet-modified protein (as well as any composition include a tet-modified protein), the proteinthat incorporates the tet-amino acid was expressed, purified, and characterized in accordance with the protocols described by WO 2016 / 176689 (PCT / US2016 / 030469). The oligonucleotide of a set length and sequence, and having a functional group on a terminal end, was purchased and a functional group on the terminal end of the oligonucleotide was converted to TCO. The tet- modified protein was conjugated to the oligonucleotide-TCO at ambient for about one hour.

[0335] For PrOCs that are multimers, such as SAPO multimers, a plurality of PrOC were formed and used as CS. A PS was purchased which included sequences complementary to the CS(s) and the PrOCs were annealed to the PS.

[0336] For PrOCs with a multimer linker, the PrOC(s) were formed as described above. A multimer linker was purchased with a set number of branches, length of branches, and optionally, with differentiated functional groups on the branches. At least one set of functional groups on the multimer linker was converted to TCO or to otherwise be configured to react with tet. In some examples, another set of the functional groups was converted to react with the oligonucleotide. The protein-tet(s) were conjugated to multimer linker via the TCO on the branch(s) and the oligonucleotide was conjugated to the oligo-functional branch of the multimer linker either simultaneously or sequentially. FIGs. 34A-34B show Tables 8 and 9 that describe the reagents and procedures for forming each of the PrOC constructs or multifluor constructs used in the experiments described above.

[0337] As shown in Table 9, the reactions that used SPAAC chemistry typically ran overnight or 20 hours. The reactions that used acid / base chemistry also ran at similar time intervals. In contrast, the IEDDA chemistry ran much faster, typically in one hour or less. In terms of reagent used, the acid / base reaction used large excess of one reagent relative to the other, the SPAAC reaction used no to small excess reagents, and the IEDDA reaction used no excess reagents. Thus, the IEDDA reaction was most efficient in terms of reagent utilization and time needed for the reaction.

[0338] As described above, various experiments included and / or used a tet- modified protein. The tet-modified protein included a substitution of an amino acid from the wild type protein with a tet-amino acid at a particular position. For example, wild type sfGFP and dGFP were modified by substituting the amino acid at position 150 with a tetrazine-amino acid (ncAA). Wt Nb22 was modified by substituting the amino acid at position 134 with a tetrazine-amino acid ,and wild type aPD-L1 VHH6 was modified by substituting the amino acid at position 131 with a tetrazine-amino acid. Other proteins that were modified included lactate oxidase, Protein A Z domain, and aA1 . The wild type (wt) sequences of the proteins along with an isolation tag (six HIS) and which leave out export sequences or solubility tags for more convenient expression are provided by SEQ ID NOs: 1-7, including wt dGFP (SEQ ID NO: 1 ), wt sfGFP (SEQ ID NO: 2), wt NB22 (SEQ ID NO: 3), wt lactate oxidase (SEQ ID NO: 4), wt Protein A Z Domain (SEQ ID NO: 5), wt aA1 (SEQ ID NO: 6), and wt aPD-L1 VHH6 (SEQ ID NO: 7). Some of the sequence include the lead MET, while others did not.

[0339] More specifically, wt dGFP (SEQ ID NO: 1 ) includes:MVSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQK NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLYTQSVLSKDP NEKRDHMVLLEFVTAAGITHGMDELYKGSHHHHHH.

[0340] Wt sfGFP (SEQ ID NO: 2) includes: MVSKGEELFTGWPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGK LPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQK NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDP NEKRDHMVLLEFVTAAGITHGMDELYKGSHHHHHH.

[0341] Wt NB22 (SEQ ID NO: 3) includes: QVQLQESGGGSVQDGGSLRLSCAASGYAYDTYYMGWFRQAPGKEREGVAG ITSLVSGVAYYKYYTDSVKGRFTIFRDDDKNTVDLQMNSLKPEDTAIYYCAASR SGLRARLLRPELYEYWGQGTQVTVSSGSGXGSGENLYFQGGSHHHHHH.

[0342] Wt lactate oxidase (SEQ ID NO: 4) includes:MGHHHHHHGENLYFQGTNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAGHSGLPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAPGSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQN VEDLKGLDLFDNPYGYEY.

[0343] Wt Protein A Z Domain (SEQ ID NO: 5) includes:MADNKFNKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGTHHHHHH.

[0344] Wt aA1 (SEQ ID NO: 6) includes:LSQVRLQESGPSLVKPSQTLSLTCTVSGFSLSSDGVGWVRQAPGKALEWVGNVFRSGATWYNPALKSRLSITRDTSTSQVSLSVSSVTTEDTAVYYCARGGAITFVDFDAWGPGLLVTVSSGGGGSGGGGSGGGGSGGGGSRAMLTQPSSVSRSLGQIVSITCSGSSRNVGFGYSVSWYQLIPGSAPRTLIYDSTSRASGVPDRFSGSRSGNTATLTISSLQPEDEAAYYCASRDITDYGVFGSGTRLTVLGSGSGSGL VPRGSHHHHHH.

[0345] Wt aPD-L1 VHH6 (SEQ ID NO: 7) includes:EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYHMAWFRQAPGKEREFVAGISWTGRGTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEGTLY GSGGRTHQSAYDYWGQGTQVTVSSGSGSGSGENLYFQGGSHHHHHH.

Claims

CLAIMS1 . A composition comprising: a protein; and an oligonucleotide linked to the protein via a tetrazine and a trans- cyclooctene (TCO) in a predetermined relationship.

2. The composition of claim 1 , wherein the protein and the oligonucleotide form a conjugate configured to bind to a target analyte.

3. The composition of claim 1 , wherein: the protein is bound to the tetrazine; and the oligonucleotide is bound to the TCO and the TCO is bound to the tetrazine.

4. The composition of any of claims 1-3, wherein the protein incorporates the tetrazine.

5. The composition of claim 1 , wherein: the protein is bound to the TCO; and the oligonucleotide is bound to the tetrazine and the TCO is bound to the tetrazine.

6. The composition of claim 1 , wherein the predetermined relationship includes a ratio of protein to oligonucleotide of p:o, p:1 , or 1 :o.

7. The composition of 6, wherein the predetermined relationship includes the ratio of protein to oligonucleotide of 1 : 1.

8. The composition of claim 6, wherein o and / or p are greater than 1 .

9. The composition of claim 8, wherein p and o are different numbers or the same number that is greater than 1.

10. The composition of claim 1 , wherein the predetermined relationship includes a ratio of protein to tetrazine of 1 :1 .11 . The composition of claim 1 , wherein the predetermined relationship includes a ratio of protein to tetrazine of 1 :n, p: 1 , or p:n, wherein n and / or p are greater than 1 .

12. The composition of claim 11 , wherein n and p are different numbers or the same number that is greater than 1 .

13. The composition of claim 1 , further including a multimer linker that links the oligonucleotide and the protein, wherein the protein is linked to the multimer linker via the tetrazine and the TCO.

14. The composition of claim 13, wherein the multimer linker comprises a plurality of branches, wherein one of the plurality of branches is linked to the oligonucleotide and a second of the plurality of branches is linked to the TCO and linked to the protein via the tetrazine and the TCO, the composition further including additional proteins linked to tetrazine and linked to the remaining plurality of branches of the multimer linker via the tetrazines and TCO.

15. The composition of claim 13, wherein the multimer linker comprises a plurality of branches, wherein one of the plurality of branches is linked to the oligonucleotide, the composition further including additional proteins linked to tetrazine and additional oligonucleotides linked to TCO and which are linked to the remaining plurality of branches of the multimer linker.

16. The composition of claim 1 , wherein the composition comprises a selfassembled protein-oligonucleotide (SAPO) multimer comprising the protein and oligonucleotide, and a second oligonucleotide.

17. The composition of claim 16, wherein the second oligonucleotide is a primary strand and the oligonucleotide is complementary to a portion of the primary strand, and the composition further including: a third oligonucleotide that is complementary to another portion of the primary strand: and a second protein linked to the third oligonucleotide via another tetrazine and TCO.

18. The composition of claim 1 , wherein the protein is a single domain antibody (sdAb).

19. The composition of claim 1 , wherein the protein is an antibody selected from a full-size antibody, an enzyme, a single variable domain heavy chain (VHH), a single-chain variable fragment (scFv) antibody, or an antibody fragment (Fab).

20. The composition of claim 1 , wherein the protein includes an antibody that includes a single domain antibody (sdAb).21 . The composition of claim 1 , wherein the protein is a non-single domain antibody (sdAb) protein, the protein optionally being an enzyme or an assay signal protein.

22. The composition of claim 1 , wherein the protein or the oligonucleotide are configured to bind to a target analyte.

23. The composition of claim 1 , wherein the oligonucleotide is single stranded and / or double stranded.

24. The composition of claim 1 , wherein the oligonucleotide includes a length of between about 2 nucleotides and about 500 nucleotides.

25. The composition of claim 1 , wherein the oligonucleotide includes a length of between about 1 nucleotide and about 100 nucleotides.

26. A kit comprising: a protein linked to a tetrazine; and an oligonucleotide linked to a trans-cyclooctene (TCO), wherein the TCO is configured to react with the tetrazine to link the oligonucleotide to the protein.

27. The kit of claim 26, wherein the TCO is configured to react with the tetrazine such that the oligonucleotide is linked to the protein via the tetrazine and the TCO in a predetermined relationship, the predetermined relationship including a ratio of protein to oligonucleotide 1 :1.

28. The kit of claim 26, wherein the TCO is configured to react with the tetrazine such that the oligonucleotide is linked to the protein via the tetrazine and the TCO in a predetermined relationship, the predetermined relationship including a ratio of protein to oligonucleotide of p:o, p:1 , or 1 :o.

29. The kit of claim 28, wherein o and / or p are greater than 1 .

30. The kit of claim 28, wherein p and o are different numbers or the same number that is greater than 1 .31 . The kit of claim 26, wherein the TCO is configured to react with the tetrazine, wherein tetrazine is in a predetermined relationship with the protein, the predetermined relationship including a ratio of protein to tetrazine of 1 :1.

32. The kit of claim 26, wherein the TCO is configured to react with the tetrazine, wherein tetrazine is in a predetermined relationship with the protein,the predetermined relationship including a ratio of protein to tetrazine of 1 :n, p:1 , or p:n, n and, optionally p, being greater than 1 .

33. The kit of claim 26, further including a multimer linker configured to bind the oligonucleotide and the protein via the tetrazine and the TCO reaction.

34. The kit of claim 33, wherein the multimer linker comprises a plurality of branches, wherein one of the plurality of branches is configured to bind to the oligonucleotide and a second of the plurality of branches is linked to the TCO and configured to bind to the protein via the tetrazine and the TCO reaction, the kit further including additional proteins linked to tetrazine and configured to bind to the remaining plurality of branches of the multimer linker.

35. The kit of claim 33, wherein the multimer linker comprises a plurality of branches, one of the plurality of branches being configured to bind to the oligonucleotide, the kit further including additional proteins linked to tetrazine and additional oligonucleotides linked to TCO and configured to bind to the remaining plurality of branches of the multimer linker.

36. The kit of claim 16, wherein the kit comprises components for a selfassembled protein-oligonucleotide (SAPO) multimer including the protein, the oligonucleotide, and a second oligonucleotide.

37. The kit of claim 36, wherein the second oligonucleotide is a primary strand and the oligonucleotide is complementary to a portion of the primary strand, and the kit further includes: a third oligonucleotide that is complementary to another portion of the primary strand; and a second protein linked to the third oligonucleotide via another tetrazine and TCO.

38. The kit of claim 26, wherein the protein incorporates the tetrazine or the tetrazine is introduced to the protein.

39. The kit of claim 26, wherein the protein is a single domain antibody (sdAb).

40. The kit of claim 26, wherein the protein is an antibody.41 . The kit of claim 40, wherein the antibody includes a single domain antibody (sdAb).

42. The kit of claim 26, wherein the oligonucleotide is single stranded and / or double stranded.

43. The kit of claim 26, wherein the oligonucleotide is deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA).

44. The kit of claim 26, further including a second oligonucleotide that is complementary to the oligonucleotide, the oligonucleotide and second oligonucleotide being single stranded.

45. The kit of claim 44, wherein the second oligonucleotide includes a first linker and the kit further including a signal construct including a second linker configured to bind with the first linker of the second oligonucleotide.

46. The kit of claim 45, wherein the signal construct includes a fluorophore, a multifluor, a dye, the second oligonucleotide, a particle, a magnetic component, or any combination thereof.

47. The kit of claim 26, wherein the TCO is configured to react with the tetrazine to form a protein-oligonucleotide conjugate.

48. A kit comprising: a conjugate including a protein conjugated to an oligonucleotide through a tetrazine and a trans-cyclooctene (TCO) in a predetermined relationship; and a second oligonucleotide that is complementary to the oligonucleotide and which forms at least part of or is to linked to a signal construct, wherein the signal construct is used to derive a detectable signal.

49. The kit of claim 48, wherein the second oligonucleotide includes or is linked to a first linker and the signal construct includes a second linker configured to react with the first linker, the second linker being bound to a signal component.

50. The kit of claim 48, wherein the predetermined relationship includes a ratio of protein to oligonucleotide of 1 :1 .51 . The kit of claim 48, wherein the predetermined relationship includes a ratio of protein to oligonucleotide of p:o, p:1 , or 1 :o, wherein o and / or p are greater than 1.

52. The kit of claim 51 , wherein p and o are different numbers or the same number that is greater than 1 .

53. The kit of claim 48, wherein the predetermined relationship includes a ratio of protein to tetrazine of 1 :1 .

54. The kit of claim 48, wherein the predetermined relationship includes a ratio of protein to tetrazine of 1 :n or p:1 , n or p being greater than 1 .

55. The kit of claim 48, wherein the predetermined relationship includes a ratio of protein to tetrazine of p:n, wherein p and n are a different number or are the same number that is greater than 1 .

56. The kit of claim 48, wherein the protein incorporates the tetrazine.

57. The kit of claim 48, wherein the protein is a single domain antibody (sdAb).

58. The kit of claim 48, wherein the protein is an antibody.

59. The kit of claim 58, wherein the antibody includes a single domain antibody (sdAb).

60. The kit of claim 48, wherein the oligonucleotide and second oligonucleotide are single stranded.61 . The kit of claim 48, wherein the oligonucleotide and second oligonucleotide are each deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA).

62. The kit of claim 48, wherein the signal construct includes a fluorophore, a multifluor, a dye, the second oligonucleotide, a particle, a magnetic component, or any combination thereof.

63. The kit of claim 48, further including a set of conjugates including the conjugate, a set of second oligonucleotides including the oligonucleotide, and a set of signal constructs including the signal construct, wherein the set of conjugates each include different proteins configured to bind to a different target analyte or a different epitope of a target analyte.

64. The kit of claim 48, wherein the signal construct includes a fluorophore, the second oligonucleotide, a particle, or a combination thereof, and is configured to output a signal derived from: a fluorescent signal, a change in mass, a change in charge, or a combination thereof.

65. A method comprising: exposing a sample to: a protein; and an oligonucleotide linked to the protein via a tetrazine and a trans- cyclooctene (TCO) in a predetermined relationship; and identifying and / or using complexes formed that include the protein or oligonucleotide bound to a target analyte, the protein being linked to the oligonucleotide via the tetrazine and TCO.

66. The method of claim 65, wherein exposing the sample includes: exposing the protein linked to the tetrazine to the sample, wherein the protein or oligonucleotide is configured to bind to a target analyte; and removing unbound protein and oligonucleotide.

67. The method of claim 65, wherein identifying conjugates formed further includes exposing the conjugates to signal constructs including a second oligonucleotide linked to a signal component via a linker, wherein the second oligonucleotide is complementary to the oligonucleotide.

68. The method of claim 67, wherein the method includes at least one of: exposing the sample to a volume of the protein linked to the oligonucleotide, wherein the protein or oligonucleotide is configured to bind to a target analyte; removing protein of the volume that is unlinked to the target analyte; exposing the sample to a volume of the signal constructs; removing second oligonucleotide unlinked to the target analyte; and identifying the complexes the signal constructs.

69. The method of claim 67, further including removing or inactivating the signal component.

70. The method of claim 65, wherein identifying complexes formed that include the protein or oligonucleotide bound to the target analyte comprises modulating a charge and / or mass of the target analyte using the protein linked to the oligonucleotide.71 . The method of claim 65, further including exposing the sample to a second oligonucleotide that is complementary to the oligonucleotide and which modulates a charge and / or mass of the target analyte.

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