A sis divalent streptavidin (SA) complex

Abstract

The present disclosure generally relates to a method for forming a cis divalent Streptavidin (SA) complex, the complex comprising a Streptavidin core and a biotin structure arranged to connect to two biotin binding sites of the Streptavidin core, wherein the biotin structure has been specifically arranged such that at least one biotin binding site on the Streptavidin core remains unoccupied. The present disclosure also relates to a corresponding method for forming a mixture comprising the cis divalent Streptavidin (SA) complex, and to a method for forming a composition comprising such a cis divalent Streptavidin (SA) complex.

Description

[0001] A CIS DIVALENT STREPTAVIDIN (SA) COMPLEX

[0002] TECHNICAL FIELD

[0003] The present disclosure generally relates to a method for forming a cis divalent Streptavidin (SA) complex, the complex comprising a Streptavidin core and a biotin structure arranged to connect to two biotin binding sites of the Streptavidin core, wherein the biotin structure has been specifically arranged such that at least one biotin binding site on the Streptavidin core remains unoccupied. The present disclosure also relates to a corresponding method for forming a mixture comprising the cis divalent Streptavidin (SA) complex, and to a method for forming a composition comprising such a cis divalent Streptavidin (SA) complex.

[0004] BACKGROUND

[0005] In immunolabeling, antibodies are used for detection of molecules in biological and non-biological samples. Antibodies are immunoglobulin (Ig) proteins that bind with high specificity through its antigen-binding site to an antigen (target molecule). Typically, the antigen is a protein, but can be any immunogenic agent such as polysaccharides, lipids, toxins etc. The part of the antigen to which the antibody binds is called epitope. Antibodies used for immunolabeling can be polyclonal or monoclonal. Polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes of one antigen, while monoclonal antibodies show specificity for a single epitope. In general, monoclonal antibodies render more specific immunolabeling signals.

[0006] Immunolabeling can either be direct or indirect. The direct method is a one- step labeling method and involves a primary antibody that is labeled with a functional element, such as a reporter molecule. The reporter molecule (label) is a molecule that can generate a signal, such as an enzyme or fluorochrome (further described below). When the labeled primary antibody is added to a sample it binds and reveals the location and / or amount of the antigen. Since the direct method utilizes only one incubation step with the sample it is simple and rapid.

[0007] The indirect method is a two-step labeling method that results in signal amplification. It involves a primary antibody (first step) that binds to the antigen in the sample and a labeled secondary antibody (second step) that binds to the bound primary antibody. Since several labeled secondary antibody molecules bind to each primary antibody molecule, the reporter signal will be amplified compared to the direct method. However, the indirect method requires one extra incubation step and the secondary antibody needs to be carefully adjusted to the primary antibody. The secondary antibody is usually raised against the Ig class of the animal species in which the primary antibody was raised. For example, if the primary antibody is a mouse IgG antibody, the secondary antibody is an anti-mouse IgG antibody. If the primary antibody is a rabbit IgG, then the secondary antibody is an antirabbit IgG etc.

[0008] Streptavidin (or other derivates of avidin) can also be used in immunolabeling with the indirect method by using biotinylated antibodies. Biotinylated antibodies are antibodies that have been conjugated with biotin molecules (usually 3-6 biotin molecules / antibody molecules). Streptavidin binds strongly to biotin and can thus be used as a secondary reagent to biotinylated antibodies.

[0009] Streptavidin is a tetrameric protein with four high affinity binding sites for biotin (binding affinity 10'14mol / 1). The protein is composed of four identical subunits. However, the subunits are configured as a dimer of two dimers so that two biotin-binding pockets are closer together on each side of the streptavidin protein. The distance between biotin pockets on the same side (cis) are approximately 2 nm and the distance between biotin pockets on opposite sides (trans) are almost the double.

[0010] In some applications when the nature of the sample is plastic, such as biotinylated proteins in the membrane of living cells and biotinylated proteins in solutions, regular streptavidin with its four biotin-binding sites will cause protein aggregation. To avoid this phenomenon, a monovalent form of streptavidin has been engineered (Howarth M et al, Nature methods, 2006). Monovalent streptavidin has only one biotin-binding site. Consequently, it can be used to target biotinylated proteins in membranes of living cells and biotinylated proteins in solution without the risk of cross-linking proteins.

[0011] The introduction of monovalent Streptavidin marked a significant advancement in applications involving single-molecule tracking and labeling biotinylated proteins in solution. However, ironically enough, for monovalent Streptavidin the solution introduces a new problem. That is, while this singular biotin-binding site avoids cross-linking and aggregation, it introduces challenges in applications where multiple reporter molecules are necessary for enhanced signal detection or when the reporter molecules are bulky.

[0012] Attaching bulky and / or multiple reporter molecules to a monovalent Streptavidin can lead to a phenomenon known as steric hindrance. Steric hindrance occurs when the physical presence of one molecule prevents the proper binding or interaction of another molecule due to spatial constraints. In the context of monovalent Streptavidin, this means that the attachment of a reporter molecule could physically block or limit access to the biotin-binding site, thereby preventing the binding of monovalent streptavidin to its biotinylated target, such a biotinylated antibody. This situation can significantly compromise the efficacy of labeling, as it may lead to weaker signals or incomplete detection of the target molecules.

[0013] However, strict monovalent Streptavidin with only one functional biotinbinding site is not always required to avoid aggregation or clumping of biotinylated molecules. A divalent Streptavidin offers a promising alternative, provided that the two functional biotin-binding sites are in a cis configuration and that the biotinylated molecules are at a certain size. Taking advantage of the size of bigger biotinylated molecules and the short distance between two biotin-binding sites in cis position, a divalent Streptavidin can also show monovalent binding properties. This is because the binding of a biotinylated molecule at a certain size to one of the two biotin-binding sites of cis divalent Streptavidin will sterically block the access of a second biotinylated molecule to the second biotin-binding site. Advantageously, because of two functional biotin-binding sites, cis divalent Streptavidin is much more resistant to losing its biotin-binding quality when conjugated with functional elements, such as reporter molecules. Thus, cis divalent Streptavidin strikes a balance between preventing aggregation and maximizing biotin binding. This divalent configuration allows for greater flexibility in labeling strategies, accommodating the attachment of bulky and multiple reporter molecules without significant risk of blocking the biotin-binding quality.

[0014] Additionally, the presently available methods for creating the desired divalent Streptavidin form, as described above (cis divalent Streptavidin), are not ideally suited for large-scale production. These methods typically involve genetically modified subunits of Streptavidin that are mixed and purified, a process that can be labor-intensive, inefficient, and inconsistent, thereby hindering industrial applicability and scalability. In contrast, a method based on conventional tetravalent Streptavidin as starting material, would benefit from the already established large-scale production of Streptavidin on the market.

[0015] With these considerations in mind, there is a clear need for a novel approach for forming cis divalent Streptavidin complexes. Such approaches should not only address the limitations associated with monovalent Streptavidin but also ensure that the process is compatible with industrial-scale production. SUMMARY

[0016] According to a first aspect of the present disclosure, the above is at least partly achieved by a cis divalent Streptavidin (SA) complex, comprising a Streptavidin core comprising four biotin binding sites, and a biotin structure comprising two biotins and a bridge linker, wherein each of the two biotins is connected to opposite sides of the bridge linker, wherein the biotin structure has a reachable length selected to be within a predefined range, the predefined range having a lower limit corresponding to the distance between biotin-binding sites in cis positions of the Streptavidin core, and the biotin structure is connected to two of the biotin-binding sites of the Streptavidin core in a cis conformation.

[0017] The present disclosure is based on the understanding that optimizing the interaction between Streptavidin and biotinylated molecules / particles in a controlled manner can significantly enhance the efficiency and specificity of binding events in biological and analytical applications. By precisely configuring a cis divalent Streptavidin (SA) complex, which incorporates a biotin structure linked by a bridge linker, the present disclosure harnesses the inherent strong affinity of Streptavidin for biotin while avoiding the limitations observed in monovalent (steric loss of biotin binding) and tetravalent forms (aggregation). The design ensures that the biotin structure engages with the Streptavidin core in a cis conformation, a strategic orientation that leverages the spatial arrangement of biotin-binding sites for optimal functionality. To be noted, within the context of the present disclosure the term Streptavidin also includes modified versions of tetravalent streptavidin, such as for example traptavedin and flavedin.

[0018] Advantages with the present disclosure include maintaining two active biotinbinding sites in a cis conformation, whereby the complex achieves a balance between minimizing potential cross-linking / aggregation, and still enabling robust, specific binding to larger biotinylated molecules, such as biotinylated proteins. This duality is desirable in applications requiring monovalent binding properties without compromising the biotinbinding efficiency. Furthermore, the specified range for the biotin structure is specifically tailored to favor the formation the desired cis configuration, enhancing the yield and stability of the cis divalent Streptavidin complex. Such a careful design consideration minimizes formation of contaminating byproducts that can arise with indiscriminate lengths of the biotin structure. Accordingly, the present disclosure not only addresses the limitations associated with existing Streptavidin modifications, but also opens up new avenues for developing more efficient, reliable, and versatile biotin-Streptavidin binding systems, in a wide range of biotechnological and diagnostic applications. The present disclosure is taking advantage of the position of the four biotinbinding pockets of Streptavidin. The four pockets are configured as two pairs of biotinbinding sites located on opposite sides of the Streptavidin molecule. The distance between two biotin-binding sites within one pair (cis position) is only 2 nm. Since they are facing the same direction, one binding site will be sterically blocked when the other binding site captures a biotinylated molecule larger than 2 nm in width. Thus, the cis divalent Streptavidin complex is divalent for small biotinylated molecules (<2 nm in width), but monovalent for larger biotinylated molecules (>2 nm in width).

[0019] Previous strategies to engineer monovalent Streptavidin have been based on making three of the four biotin-binding non-functional, leaving only one site open for biotinbinding. Such variants of monovalent Streptavidin are monovalent for all biotinylated molecules, but the single biotin-binding site makes them vulnerable for steric hindrance when conjugated with function elements. The present disclosure defines a different Streptavidin complex with monovalent binding quality to larger biotinylated molecules and enhanced biotin-binding capacity that better resists steric hindrance, when conjugated with functional elements. Examples of larger molecules (>2 nm in width) suitable for monovalent binding by the cis divalent Streptavidin complex are biotinylated proteins, including antibodies and antigens, biotinylated liposomes, biotinylated Virus Like Particles (VLP), biotinylated nanoparticles, including metal nanoparticles, polymeric nanoparticles, ceramic nanoparticles, and lipid nanoparticles.

[0020] It is in accordance with the present disclosure preferred to also introduce an upper limit of the predefined range of the biotin structure. Generally, the upper limit of the range is selected to minimize biotin binding in trans positions of the Streptavidin core. In optimizing the cis divalent Streptavidin (SA) complex, the upper limit of the predefined range of the biotin structure is used for fine-tuning the system’s specificity and efficiency. By selecting an upper limit that minimizes biotin-binding in trans positions of the Streptavidin core, such an approach ensures that the biotin structures are primarily oriented in the desired cis conformation. The orientation is specifically desirable in preventing the potential aggregation or nonspecific interactions that might occur if biotin molecules were to bind indiscriminately to any available binding sites, including those in trans positions.

[0021] In an embodiment, the reachable length of the biotin structure is controlled by selecting a length of the bridge linker to be from 2 to 5 nm, preferably from 2 to 4 nm. The distance between trans positions of the Streptavidin core is close to 4 nm, making this range particularly relevant for ensuring that the biotin structures do not inadvertently promote binding configurations that could detract from the complex’s intended specificity and performance. The lower limit is the distance between two biotin-binding pockets in cis conformation.

[0022] The precision in defining the upper limit of the biotin structure serves not only to enhance the binding specificity of the cis divalent Streptavidin (SA) complex but also to optimize its functional utility in a broad range of applications, from biochemical assays to therapeutic delivery systems.

[0023] It should be noted that, if the biotin structure needs to be fully stretched to reach the biotin-binding pockets in cis position, it will start to favor biotin-binding also between two neighboring Streptavidin molecules and thus initiate cross-linking. Therefore, a slightly longer biotin structure than 2 nm will be beneficial. For the upper limit, the distance between two biotin-binding pockets in trans conformation is important. A too long biotin structure will also reach the biotin-binding pockets in trans positions, resulting in formation of trans divalent Streptavidin byproducts. Such byproduct populations may be cleaned up using purification methods. However, over time some biotins in cis position can detach and re-attach in trans position and thereby spontaneously form new impurities in purified cis divalent Streptavidin sample.

[0024] In a possible embodiment of the present disclosure, the bridge linker comprises at least one structure selected from the group consisting of amino acids, nucleotides, carbohydrates, polyethylene glycol, polypropylene glycol, polylactic acid, and polycaprolactone, 2,2-bis(methylol)propionic acid and / or a rigid structure component selected from the group consisting of aromatic groups, heteroaromatic groups, double bond systems, and rigid cyclic structures. Other structures having similar characteristics are of course possible and within the scope of the present disclosure.

[0025] Preferably, the cis divalent Streptavidin (SA) complex further comprises a functional element arranged to label the cis divalent Streptavidin (SA) complex. For example, the functional element may include one or a plurality of a DNA oligonucleotide (single or double stranded), a RNA oligonucleotide (single or double stranded), a protein, a peptide, an enzyme, a carbohydrate structure, a nanoparticle, a hapten / antigen, a fluorochrome, a dendron, a dendrimer, a polyethylene glycol (single-armed or multi-armed), quantum dots, metal ion, a maleimide, a thiol group, an azide, an alkyne group (and variants of alkynes used for click chemistry with azide), a SpyTag, a SpyCatcher, and a therapeutic agent. Some of the disclosed functional elements are known to the skilled person, it should however be understood that other / future reporter elements and attachments elements may equally be used in relation to the present disclosure.

[0026] In an embodiment of the present disclosure, the functional element is conjugated to the Streptavidin core. This could for example be performed by an ester reaction to amino groups in the Streptavidin core protein (N-terminus of each Streptavidin subunit and side chain of lysin residues). However, the exact location of the conjugated functional elements cannot be fully controlled with such a method. For more precise conjugation, variants of genetically engineered Streptavidins, such as flavidin can be used.

[0027] Advantageously, the bridge linker comprises at least one bridge linker conjugation site. This locates conjugation of functional elements to the biotin structure on the non-binding side of the cis divalent Streptavidin complex. This embodiment of the present disclosure divides the cis divalent Streptavidin complex into one biotin-binding side and one conjugation side for functional elements. This minimizes the risk of functional elements to interfere with the biotin-binding property of Streptavidin, a feature that is highly important when bulky and / or many functional elements are conjugated to Streptavidin.

[0028] In one embodiment of the present disclosure the biotin structure contains one single conjugation site for functional elements. This creates a version of the complex that is highly suited for methods that require a precise 1 : 1 ratio of the number of functional elements to the cis divalent Streptavidin complex. This complex will have implications in quantitative streptavidin-based assays.

[0029] It should be understood that the functional element alternatively or also may be conjugated to the at least one bridge linker conjugation site.

[0030] Furthermore, it should be understood that it may be possible to allow the conjugation of the functional element to the bridge linker to be facilitated by a click chemistry reaction.

[0031] Furthermore, functional elements can simultaneously be conjugated to both the Streptavidin core protein and the biotin structure. The functional elements could either be the same on the Streptavidin core protein and the biotin structure, or they could be different. The latter could for example be two different fluorescent dyes, or two different attachment elements such as azide for click chemistry and maleimide for attachment of thiol groups.

[0032] Furthermore, the biotin structure could be synthesized to contain two different functional elements, preferably with high stoichiometric precision. In one embodiment of the present disclosure, the biotin structure contains two different reactive groups for use with two different click chemistry reactions. This could for example be a first reactive group for Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC), such as a terminal alkyne, and a second reactive group for Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC), such as dibenzocyclooctyne (DBCO) or a tetrazine. Taking advantage of the different reaction conditions for CuAAC and SPAAC, different molecules can be conjugated to the first and second reactive groups on the biotin structure using click chemistry.

[0033] It should be understood that a first reactive group for click chemistry could also be attached to the Streptavidin core and the second reactive group for click chemistry to the biotin structure.

[0034] According to second aspect of the present disclosure there is provided a method for forming a mixture comprising a cis divalent Streptavidin (SA) complex, wherein the method comprises the steps of providing a Streptavidin core comprising four biotin binding sites, providing a biotin structure comprising two biotins and a bridge linker, wherein each of the two biotins is connected to opposite sides of the bridge linker and a reachable length of the biotin structure selected to be within a predefined range, the predefined range having a lower limit corresponding to the distance between biotin-binding sites in cis positions of the Streptavidin core, and forming, in a reaction vessel, the mixture by reacting the Streptavidin core with the biotin structure.

[0035] Mixing the biotin structure with the Streptavidin core protein will result in a mixture of products, including Streptavidin core proteins (tetravalent), cis divalent Streptavidin complexes, two versions of trans divalent Streptavidin complexes, non-binding Streptavidin complexes with two biotin structures attached, and cross-linked Streptavidin where the biotin structure bind two Streptavidin core proteins. Several parameters are needed to be considered to obtain a high yield of the desired cis divalent Streptavidin (SA) complex. As discussed above, it is important to restrict the length of bridge linker to minimize the formation of trans divalent Streptavidin (SA) complexes. Conversely, the bridge linker needs to be long enough to easily reach the two biotin pockets in cis position. A too “tight” bridge linker will start to promote cross-linking of Streptavidin instead of forming cis divalent Streptavidin (SA) complexes.

[0036] Preferably, the method according to the present disclosure further comprises the step of purifying the cis divalent Streptavidin (SA) complex from the mixture using a predefined purification scheme. A pure population of cis divalent Streptavidin (SA) complex is important for its application in quantitative analytical methods. A purification step would also except a higher degree of byproducts to be formed when mixing Streptavidin core protein and biotin structure in the reaction vessel. Advantageously, the predefined purification scheme is selected to be at least one of affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, centrifugation, and filtration.

[0037] In an embodiment, the method further comprises conjugating a functional element to the cis divalent Streptavidin (SA) complex, wherein the functional element is arranged to label the cis divalent Streptavidin (SA) complex. The conjugation may in some implementations take place post purification, however it may also be possible to conjugate the functional element prior to the purification step.

[0038] As an example, in a preferred embodiment the biotin structure contains a tag or a functional element that facilitates the purification steps by altering the properties of the cis divalent Streptavidin complex compared to the Streptavidin core protein. Such properties could for example be increase of electric charge, increase of hydrophobicity / hydrophilicity, and increase in size. The tag / functional element could also be used for affinity chromatography by providing a His-tag, oligonucleotide sequence, an antigen, or other suitable structure for affinity binding. This embodiment provides advantages in yield and purity when purifying the cis divalent Streptavidin complex.

[0039] According to a third aspect of the present disclosure, and as generally discussed above, there is provided a method for forming an immunolabeling composition, wherein the method comprises selecting a biotinylated primary antibody, selecting a cis divalent Streptavidin (SA) complex as discussed above, and mixing, in a reaction vessel, the biotinylated antibody with the cis divalent Streptavidin (SA) complex to form the immunolabeling composition. In one embodiment, the cis divalent Streptavidin complex can be utilized for interaction with biotinylated antibodies in solution without inducing aggregation or cross-linking. Antibodies are three times larger than Streptavidin and their thinnest part is 3-4 nm in width. Due to the significant size of antibodies, the cis divalent Streptavidin complex will only fit the binding of one biotinylated antibody, despite possessing two biotin-binding pockets. Thus, although the cis-divalent Streptavidin complex features two biotin binding sites, its binding interaction with biotinylated antibodies remains monovalent. As generally discussed above, when conjugated with functional elements, the cis divalent Streptavidin is more resistant to lose its biotin-binding capacity compared to monovalent Streptavidin with only one biotin-binding site. This is of extra importance for bulky functional elements, such as nanoparticles, oligonucleotides, enzymes, peptides, and protein-based fluorophores. According to a fourth aspect of the present disclosure, there is provided a method for forming a composition, comprising selecting a biotinylated molecule, selecting a cis divalent Streptavidin (SA) complex, the cis divalent SA complex comprising a Streptavidin core having four biotin-binding sites, wherein a biotin structure comprising two biotins connected to opposite ends of a bridge linker is bound to two biotin-binding sites in cis on the Streptavidin core, and mixing, in a reaction vessel, the biotinylated molecule with the cis divalent SA complex to bind the biotinylated molecule to one of the two remaining biotin-binding sites of the Streptavidin core unoccupied by the biotin structure, wherein the biotinylated molecule has a size selected to sterically hinder binding of another biotinylated molecule to the remaining unoccupied biotin-binding site of the Streptavidin core. This aspect provides generally similar advantages as discussed in relation to the previous aspects of the present disclosure.

[0040] In an embodiment, the biotin structure is arranged to have a reachable length selected to be within a predefined range, the predefined range having a lower limit corresponding to the distance between biotin-binding sites in cis positions of the Streptavidin core.

[0041] It should be understood that the cis divalent Streptavidin (SA) complex selected for use in relation to the present composition may be arranged to have any and all of the further functions and features as discussed in relation to the first and the second aspect of the present disclosure, and also aligns with the third aspect as elaborated above.

[0042] Preferably, the biotinylated molecule is selected to have a width greater than or equal to 2 nm. In some embodiments, the biotinylated molecule has a width in the range of 2 to 300 nm, more preferably 2.5 to 50 nm. Such a range is suitable for many biologically relevant molecules, such as antibodies, antigens, enzymes, nucleic acid-protein complexes, and various nanoparticles. Molecules within this range are sufficiently large to sterically hinder binding of a second biotinylated molecule to the remaining biotin-binding site of the cis divalent Streptavidin complex.

[0043] By selecting such a molecular size and or range of sizes, the bound biotinylated molecule will sterically block access to the second unoccupied biotin-binding site located in cis on the same side of the Streptavidin core. The steric blocking effect as is provided by means of the present disclosure prevents the binding of additional biotinylated molecules, thereby ensuring monovalent functionalization even though the complex retains two available biotin-binding sites. Such a mechanism is particularly advantageous when working with larger biomolecules or nanoparticles, where uncontrolled multivalent binding could lead to cross-linking, aggregation, or reduced performance in downstream applications.

[0044] Still further, in accordance with the present disclosure, the biotinylated molecule is selected from the group consisting of a biotinylated protein, a biotinylated nanoparticle, such as a lipid, metal, polymer or ceramic nanoparticle, a biotinylated virus like particle (VLP), and a biotinylated polymer. For example, the biotinylated protein may be an antibody or an antigen.

[0045] Further features of, and advantages with, the present disclosure will become apparent when studying the appended claims and the following description. The skilled addressee realize that different features of the present disclosure may be combined to create embodiments other than those described in the following, without departing from the scope of the present disclosure.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The various aspects of the present disclosure, including its particular features and advantages, will be readily understood from the following detailed description and the accompanying drawings, in which:

[0048] Figs. 1 A and IB shows currently preferred embodiment of cis divalent Streptavidin (SA) complexes according to the present disclosure,

[0049] Figs. 2A - 2D illustrates exemplary steps for forming the presents steps for forming the cis divalent Streptavidin (SA) complex as presented in Fig. 1 A,

[0050] Fig. 3 presents the cis divalent Streptavidin (SA) shown in Fig. IB conjugated with an antibody, and

[0051] Fig. 4 is a flowchart exemplifying a method for forming an immunolabeling composition, comprising the cis divalent Streptavidin (SA) complex as presented in Fig. 1 A.

[0052] DETAILED DESCRIPTION

[0053] The present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which currently preferred embodiments of the present disclosure are shown. This present disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the present disclosure to the skilled addressee. Like reference characters refer to like elements throughout. Referring now to the drawings and to Fig. 1 A in particular, showing a currently preferred embodiment of a cis divalent Streptavidin (SA) complex 100 according to the present disclosure. A scheme for forming the cis divalent Streptavidin (SA) complex 100 will be further elaborated below in relation to Figs. 2A - 2B.

[0054] As presented in Fig. 1A, the cis divalent Streptavidin (SA) complex 100 comprises a Streptavidin core 102, in turn comprising four biotin binding sites 104. To the Streptavidin core 102, the cis divalent Streptavidin (SA) complex 100 incorporates a biotin structure (202 as discussed below in relation to e.g. Fig. 2A). This biotin structure is consisting of two biotin molecules 106 that are positioned on opposite sides of a bridge linker 108. The configuration of the biotin structure is such that it aligns with the biotin-binding sites 104 in cis positions on the Streptavidin core 102, with the intention to form a stable connection in a cis conformation.

[0055] The key to the effective functioning of the cis divalent Streptavidin (SA) complex 100 lies in the characteristics of the biotin structure. This biotin structure has a reachable length that is selected to fall within a predefined range. The lower limit of this range is congruent with the distance between biotin-binding sites in cis positions 104 of the Streptavidin core 102, ensuring a fit of both biotins 106. The upper limit of this range is selected so that the biotin structure is not able to reach biotin-binding sites in trans positions (104 and 104’) of the Streptavidin core 102.

[0056] In the formation of the cis divalent Streptavidin (SA) complex 100, the interplay between the biotin structure and the Streptavidin core 102 has been arranged to ensure that the biotin molecules 106, through the medium of the bridge linker 108, are positioned to engage the Streptavidin core 102. A precise orientation in cis position not only provides two unoccupied biotin binding sites for enhanced monovalent binding, but also contributes to the stability and functionality of the complex in various applications, ranging from diagnostic assays to targeted therapeutic delivery systems.

[0057] Fig. IB presents a further extended version 100’ of the cis divalent Streptavidin (SA) complex 100 as presented in Fig. 1 A. Specifically, the cis divalent Streptavidin (SA) complex 100’ of Fig. IB additionally comprises a bridge linker conjugation site 112 of the bridge linker 108. The bridge linker conjugation site 112 is further provided with a functional element 114, where the functional element 114 for example may be selected from a group comprising at least one of a DNA oligonucleotide, a RNA oligonucleotide, a protein, a peptide, an enzyme, a carbohydrate structure, a nanoparticle, a hapten / antigen, a fluorochrome, a dendron, a dendrimer, a polyethylene glycol (single-armed or multi-armed), quantum dots, metal ion, a maleimide, a thiol group, a reactive group for click chemistry (such as azide, alkyne, DBCO), a SpyTag, a SpyCatcher, and a therapeutic agent. Using the biotin structure for conjugation of functional elements advantageously divides the cis divalent Streptavidin (SA) complex into one side for conjugation of functional elements and one opposite side for biotin binding. This configuration creates a monovalently binding streptavidin that can be conjugated with functional elements without compromising with the biotin binding ability.

[0058] Turning now to Figs. 2A - 2B in conjunction with Fig. 4, exemplifying the procedure for forming the cis divalent Streptavidin (SA) complex, as for example exemplified in Fig. 1 A.

[0059] Fig. 2A shows the start of the process, initiated by providing, SI, the Streptavidin core 102, as discussed above, comprising the four biotin binding sites 104, and providing a biotin structure 202 comprising two biotins 106 and a bridge linker 108, and in a reaction vessel, mixing the two components to react and form products.

[0060] Fig. 2B presents different products that can be formed in the reaction vessel, including the desired cis divalent Streptavidin (SA) complex 100, unreactive Streptavidin core 102, Streptavidin core with two biotin structures 204, and cross-linked Streptavidin core 206. In this example, the biotin structure is too short to reach biotin binding sites in trans positions.

[0061] Formation of Streptavidin core with two biotin structures 204 can be reduced by using a surplus of Streptavidin core 102 over biotin structure 202 in the reaction vessel. The mixing procedure also needs to be optimized to avoid high local concentrations of biotin structures 202 in the reaction vessel. This can be for example be obtained by slowly adding the biotin structure 202 solution to the Streptavidin core 102 solution in the reaction vessel under constant mixing.

[0062] Cross-linking can be reduced by diluting each reactant (Streptavidin core 102 solution and biotin structure 202 solution) before mixing in the reaction vessel.

[0063] Turning now to Figs. 2C - 2D in conjunction with Fig. 4, exemplifying purification procedures for purifying the cis divalent Streptavidin (SA) complex 100 from other products present in the mixture exemplified in Fig. 2B, using a predefined purification scheme.

[0064] In Fig. 2C, a charged tag 208 is introduced in the biotin structure 202. The extra charge carried by the biotin structure is used for ion exchange chromatography to separate the cis divalent Streptavidin (SA) complex 100” (one charged tag) from unreactive Streptavidin core 102 (no charged tag) and Streptavidin core with two biotin structures 204’ (two charged tags). The charged tag 208 can either be negatively charged or positively charged. However, a net positive charge of the cis divalent Streptavidin (SA) complex 100” can cause unwanted unspecific binding to negatively charged extracellular matrix and DNA if used for immunolabeling of tissues for microscopy. In some embodiments, the functional element 114 is charged and used for separation of products with ion exchange chromatography. This could for example be negatively charged oligonucleotides.

[0065] The tag / functional element could also be designed to be used for affinity chromatography. This could for example be a His-tag, oligonucleotide sequence, or an antigen.

[0066] In Fig. 2D, the cis divalent Streptavidin (SA) complex 100 is purified from cross-linked Streptavidin core 206 by size. This could be performed by size exclusion chromatography where the smaller cis divalent Streptavidin (SA) complex 100 runs slower through the column than the larger cross-linked Streptavidin core 206. Purification by size could also be performed by spin column centrifugation (centrifugation / filtration) with a cutoff filter membrane that prevents the larger cross-linked Streptavidin core 206, but not the smaller cis divalent Streptavidin (SA) complex 100, to go through the membrane.

[0067] Finally, in Fig. 3, there is presented an exemplary immunolabeling composition comprising the cis divalent Streptavidin (SA) complex 100’ as presented in Fig. IB, and an antibody 302 conjugated with biotin molecules (106’). The immunolabeling complex is readily formed by mixing the cis divalent Streptavidin (SA) complex 100’ and the biotinylated antibody in a reaction vessel. In general, one biotinylated antibody carries 3-5 biotin molecules 106’ and thus has the capacity to bind several cis divalent Streptavidin (SA) complexes. To avoid steric hindrance of the antigen binding sites 304 by the cis divalent Streptavidin (SA) complex 100’, the antibody 302 should preferably not carry more than three cis divalent Streptavidin (SA) complexes 100”. To obtain a homogenous distribution of bound cis divalent Streptavidin (SA) complexes to the antibodies, the mixing procedure needs to be fast to rapidly achieve a homogenous solution of antibodies and cis divalent Streptavidin (SA) complexes in the reaction vessel. Fig. 4 shows a scheme of the procedure to produce purified cis divalent Streptavidin SA complex. The procedure starts by providing, SI, the Streptavidin core 102 in solution at a chosen concentration that minimizes crosslinking, and providing, S2, the biotin structure 202 with a selected bridge linker that minimizes biotin binding in trans positions and maximizes biotin binding in cis position S2. The biotin structure 202 solution is mixed, S3, with the Streptavidin core 102 solution to react in a reaction vessel and form the cis divalent Streptavidin (SA) complex. The cis divalent Streptavidin (SA) complex is finally purified, S4, from byproducts in the mixture with purification techniques.

[0068] Although the figures may show a specific order of method steps, the order of the steps may differ from what is depicted. Also, two or more steps may be performed concurrently or with partial concurrence. Such variation will depend on designer choice. All such variations are within the scope of the disclosure. Additionally, even though the present disclosure has been described with reference to specific exemplifying embodiments thereof, many different alterations, modifications and the like will become apparent for those skilled in the art. Variations to the disclosed embodiments can be understood and effected by the skilled addressee in practicing the claimed present disclosure, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the word ’’comprising” does not exclude other elements or steps, and the indefinite article ”a” or ”an” does not exclude a plurality.

Claims

CLAIMS1. A cis divalent Streptavidin (SA) complex, comprising: a Streptavidin core comprising four biotin binding sites, and a biotin structure comprising two biotins and a bridge linker, wherein each of the two biotins is connected to opposite sides of the bridge linker, wherein: the biotin structure has a reachable length selected to be within a predefined range, the predefined range having a lower limit corresponding to the distance between biotin-binding sites in cis positions of the Streptavidin core, and the biotin structure is connected to two of the biotin-binding sites of the Streptavidin core in a cis conformation.

2. The cis divalent Streptavidin (SA) complex according to claim 1, wherein an upper limit of the predefined range is selected to minimize biotin-binding in trans positions of the Streptavidin core.

3. The cis divalent Streptavidin (SA) complex according to claim 1, wherein an upper limit of the predefined range is selected to be less than the distance between biotinbinding sites in trans positions of the Streptavidin core.

4. The cis divalent Streptavidin (SA) complex according to claim 1, wherein a length of the bridge linker is selected to be from 2 to 5 nm, preferably from 2 to 4 nm.

5. The cis divalent Streptavidin (SA) complex according to any one of the preceding claims, wherein two of the four biotin binding sites on the Streptavidin core are occupied by the biotin structure remains, and the remaining two biotin-binding sites are unoccupied.

6. The cis divalent Streptavidin (SA) complex according to any one of the preceding claims, wherein the bridge linker comprises at least one of: amino acids, nucleotides, carbohydrates, polyethylene glycol, polypropylene glycol, polylactic acid, and polycaprolactone, 2,2-bis(methylol)propionic acid and / or a rigidstructure component selected from the group consisting of aromatic groups, heteroaromatic groups, double bond systems, and rigid cyclic structures.

7. The cis divalent Streptavidin (SA) complex according to any one of the preceding claims, further comprising a functional element arranged to label the cis divalent Streptavidin (SA) complex.

8. The cis divalent Streptavidin (SA) complex according to any one of the preceding claims, wherein the bridge linker comprises at least one bridge linker conjugation site.

9. The cis divalent Streptavidin (SA) complex according to claim 7, wherein the functional element is conjugated to the Streptavidin core.

10. The cis divalent Streptavidin (SA) complex according to claim 8 when dependent on claim 7, wherein the functional element is conjugated to the at least one bridge linker conjugation site.

11. The cis divalent Streptavidin (SA) complex according to any one of claims 7, 9 or 10, wherein the functional element is at least one of a DNA oligonucleotide, a RNA oligonucleotide, a protein, a peptide, an enzyme, a carbohydrate structure, a nanoparticle, a hapten, an antigen, a fluorochrome, a dendron, a dendrimer, a polyethylene glycol, quantum dots, metal ion, a maleimide, a thiol group, a reactive group for click chemistry, a SpyTag, a SpyCatcher, and a therapeutic agent.

12. The cis divalent Streptavidin (SA) complex according to claim 10, wherein the conjugation of the functional element to the bridge linker is facilitated by a click chemistry reaction.

13. A method for forming a mixture comprising a cis divalent Streptavidin (SA) complex, wherein the method comprises the steps of: providing a Streptavidin core comprising four biotin binding sites; providing a biotin structure comprising two biotins and a bridge linker, wherein each of the two biotins is connected to opposite sides of the bridge linker and areachable length of the biotin structure selected to be within a predefined range, the predefined range having a lower limit corresponding to the distance between biotin-binding sites in cis positions of the Streptavidin core, and forming, in a reaction vessel, the mixture by reacting the Streptavidin core with the biotin structure.

14. The method according to claim 13, wherein an upper limit of the predefined range is at least one of: selected to minimize biotin binding in trans positions of the Streptavidin core, and selected to be less than the distance between trans positions of the Streptavidin core.

15. The method according to any one of claims 13 - 14, further comprising the step of: purifying the cis divalent Streptavidin (SA) complex from the mixture using a predefined purification scheme.

16. The method according to claim 15, wherein the predefined purification scheme is selected to be at least one of affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, centrifugation, and filtration.

17. The method according to any one of claims 13 - 16, further comprising the step of: conjugating a functional element to the cis divalent Streptavidin (SA) complex, wherein the functional element is arranged to label the cis divalent Streptavidin (SA) complex.

18. The method according to claim 17, wherein the functional element is conjugated to at least one of: the Streptavidin core, and at least one bridge linker conjugation site comprised with the bridge linker.

19. The method according to any one of claims 17 and 18, wherein the functional element is selected from the group consisting of a reactive group for click chemistry, a maleimide, a thiol, DNA oligonucleotide, a protein, a peptide, an enzyme, a fluorochrome, a dendron, a dendrimer, a polyethylene glycol, and a therapeutic agent.

20. The method according to any one of claims 15 and 16, wherein the biotin structure comprises a tag or a functional element selected to facilitate the purification steps by altering properties of the cis divalent Streptavidin complex as compared to the Streptavidin core protein.

21. A method for forming an immunolabeling composition, wherein the method comprises:- selecting a biotinylated antibody;- selecting a sis divalent Streptavidin (SA) complex according to any one of claims 1 - 12, and- mixing, in a reaction vessel, the biotinylated antibody with the sis divalent Streptavidin (SA) complex to form the immunolabeling composition.

22. A method for forming a composition, comprising: selecting a biotinylated molecule, selecting a cis divalent Streptavidin (SA) complex, the cis divalent SA complex comprising a Streptavidin core having four biotin-binding sites, wherein a biotin structure comprising two biotins connected to opposite ends of a bridge linker is bound to two biotin-binding sites in cis on the Streptavidin core, and mixing, in a reaction vessel, the biotinylated molecule with the cis divalent SA complex to bind the biotinylated molecule to one of the two remaining biotin-binding sites of the Streptavidin core unoccupied by the biotin structure, wherein the biotinylated molecule has a size selected to sterically hinder binding of another biotinylated molecule to the remaining unoccupied biotin-binding site of the Streptavidin core.

23. The method according to claim 22, wherein the biotin structure has a reachable length selected to be within a predefined range, the predefined range having alower limit corresponding to the distance between biotin-binding sites in cis positions of the Streptavidin core.

24. The method according to any one of claims 22 and 23, wherein the biotinylated molecule is selected to have a width greater than 2 nm.

25. The method according to any one of claims 22 - 24, wherein the biotinylated molecule is selected from the group consisting of a biotinylated protein, a biotinylated nanoparticle, a biotinylated virus like particle (VLP), and a biotinylated polymer.

26. The method according to any one of claims 22 - 25, wherein the biotin structure comprises two different reactive groups, each arranged for conjugation to a different functional element via different attachment chemistry reactions.

27. The method according to claim 26, wherein the two reactive groups comprise a first reactive group selected from a terminal alkyne for Copper-catalyzed Azide- Alkyne Cycloaddition (CuAAC), and a second reactive group selected from a dibenzocyclooctyne (DBCO) or a tetrazine for Strain-Promoted Azide- Alkyne Cycloaddition (SPAAC).

28. The method according to any one of claims 22 - 27, wherein a first reactive group for click chemistry is attached to the Streptavidin core and a second reactive group is attached to the biotin structure.

Images