Solulink Bioconjugation PrimerJanuary 2013

Solulink Bioconjugation Primer

Introduction: Bioconjugation is the linking of two biomolecules to form a hybrid, the bioconjugate, which retains the properties of each individual component, yet yielding a single entity with two complementary functions. Biomolecules exist and function in aqueous environments; therefore, the preparation of bioconjugates is primarily about chemistry in water. Any suitable bioconjugate chemistry must be compatible with such an environment, while at the same time preserving the biological activity or function of the biomolecules. Conjugates are generally formed through the addition of separate but reactively complementary functional groups to each of the two biomolecules. The two modified biomolecules are then mixed together to form the desired bioconjugate.
Conjugation and immobilization of biomolecules has historically been very problematic for a variety of reasons; primarily because there are few covalent bond-forming reactions that proceed in water that can be engineered to link biomolecules together. A wide variety of methods to conjugate and immobilize biomolecules are extensively described in Bioconjugate Techniques, but most of these methods are difficult to perform, stoichiometrically inefficient, and result in low conjugate yields. It is important to understand the desired characteristics of the ideal bioconjugation chemistry. The following list presents many criteria that need to be fulfilled to produce the ideal bioconjugation technology:

Figure 1. Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 2008.

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a)

Linkers must be incorporated on biomolecules in a mild, controllable manner

The classical method used to prepare covalent conjugates makes use of the maleimido/thiol coupling pair. Disadvantages of the maleimido/thiol pair include:

b)

The inherent biological function of the biomolecules must be unaffected after modification and conjugation

a) c) The conjugation reaction occurs directly upon mixing the two modified biomolecules, preferably not requiring addition of an oxidant, reductant, or metal d) e) Modified biomolecules are stable over extended periods Formation of the covalent linkage is stable under a broad pH range and at elevated temperatures f) Quantification of both incorporated linkers and final conjugate is readily performed, i.e, they are spectrophotometrically traceable f) g) Conjugation occurs in buffered aqueous solutions, at a physiological pH g) h) i) j) k) l) Stoichiometrically efficient (e.g., 1:1) Fast reaction kinetics No undesirable covalent side reactions during modification h) e) b) c) d)

The aqueous instability (hydrolysis) of the linker functional group (i.e., maleimide) on biomolecules during conjugation The need to protect the thiol group The slow kinetics of the reaction The need to activate the protected thiol group with a strong reducing agent (e.g., DTT or TCEP) The need to activate the protected thiol group by addition of a strong reducing agent (e.g., DTT) The potential for undesirable homo-dimers via disulfide bridge formation Cleavage and/or partial fragmentation of proteins by reduction of disulfide bonds Reduced or inactive protein function

Next

Linkers can be incorporated on a variety of biomolecules, including oligos and peptides, through solid phase synthesis

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No electrostatic/hydrophobic interactions

The solution...

Solulink Bioconjugation PrimerJanuary 2013

The Solulink Solution

Solulink has developed a bioconjugate chemistry that more mildly, more efficiently, and more reproducibly conjugates and immobilizes proteins, peptides, oligonucleotides, carbohydrates, and polymers. This chemistry is based on the reaction of an aromatic hydrazine with an aromatic aldehyde, yielding a stable bisarylhydrazone conjugate bond (Figure 2). The aromatic hydrazine is based on 6-hydrazino-nicotinic acid (HyNic) and is incorporated on amine-containing biomolecules using S-HyNic . S-HyNic possesses a succinimidyl ester (NHS) functionality that readily reacts with amino moieties on proteins and other amine-containing biomolecules, polymers, and surfaces using standard NHS ester reaction conditions, i.e., phosphate buffer, pH 7.28.0. The aromatic aldehyde is incorporated on biomolecules with S-4FB (Figure 2). Simple addition of a HyNic-modified biomolecule to a 4FBmodified biomolecule in a mildly acidic buffer, i.e., pH 5.06.0, yields the desired conjugate. The hydrazine function is protected as its acetone hydrazone is in equilibrium with the free reactive hydrazine in aqueous solution; therefore, the HyNic group is free to react with aromatic aldehydes without a separate deprotection step, yielding the stable bisarylhydrazone. The bis-arylhydrazone has been demonstrated to be both heat stable and pH stable (95OC for 2 hr, pH 210). Solulink has also synthesized a related family of HyNic and 4FB linkers that further extend the versatility of this technology; including linkers that react with thiol moieties, contain extended hydrophilic linkers (PEG4), or are cleavable as disulfide linkers. Furthermore, HyNic moieties are readily incorporated on peptides during solid phase peptide synthesis and 4FB moieties are readily incorporated on oligonucleotides during solid phase oligonucleotide synthesis using a phosphoramidite. Another extremely useful characteristic of the bis-arylhydrazone bond is that it forms a traceable chromophore. This
Figure 2. Solulinks conjugation workflow.

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chromophore absorbs at 354 nm with a molar extinction coefficient of 29,000 (Figure 1 inset). This unique property allows the researcher to (1) follow the linking or labeling reactions in real time on a spectrophotometer, (2) directly quantify the number of linkages formed in the conjugate, and (3) visualize or trace conjugate fractions during FPLC or HPLC purification. Further increasing the versatility of the HyNic/4FB couple for the preparation of bioconjugates is the discovery by Dirksen et al.1,2 that aniline significantly catalyzes the formation of the bis-arylhdyrazone bond (Figure 3, with permission). The striking increase in conjugate reaction rate is particularly important for the conjugation of two proteins. As demonstrated in Figure 3, the conjugation of HyNic-IgG (1 mol. equiv.) to 4FB-BSA (1.5 mol. equiv.) proceeds to nearly complete conversion to conjugate in 2 hr in the presence of 10 mM aniline (Solulinks TurboLink Buffer). Maleimido/thiolbased chemistry converts only approximately 50% of IgG to conjugate when a 1:4 IgG:BSA ratio is used. The efficiency of the HyNic/4FB couple results in easier purification, significant cost savings, and better reproducibility. Also to be noted is that the efficiency of conjugation of proteins using maleimido/thiol chemistry is also dependent on the molecular weights of the two proteins. For example, conjugation of IgG (MW 150 kDa) to HRP (MW 45 kDa) proceeds more efficiently than conjugation of IgG to phycoerythrin (R-PE; MW 250 kDa). Any two proteins, regardless of molecular weights, can be efficiently conjugated using the HyNic/4FB couple in the presence of 10 mM aniline.
Figure 3. The HyNic/4FB conjugation couple. Ligation of HyNic and 4FB with aniline-catalyzed hydrazone ligation between 6-hydrazinopyridyl (HyNic)-functionalized peptide 4 and benzaldehyde 5 (left) followed by UV-VIS spectroscopy (340 nm).

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Solulink Bioconjugation PrimerJanuary 2013

HyNic/4FB Conjugation Couple Advantages

The many significant advantages to Solulinks HyNic/4FBbased conjugation technology include: a) b) c) d) e) An efficient conjugation chemistry No metals, reducing, or oxidizing agents are required Fast conjugation kinetics catalyzed by aniline Conjugate bond is stable to extremes of heat and pH Conjugate bond forms a chromophore that can be used to follow reactions in real time and quantify the number of linkages in a conjugate f) Versatile: proteins, oligos, and peptides can be rapidly conjugated to each other and to surfaces g) Direct conjugate bond formation under mild buffer conditions h) Excellent retention of inherent biological function of biomolecules following controlled modification and conjugation i) 4FB linker can be incorporated on oligonucleotides during solid phase synthesis j) HyNic linker can be incorporated on peptides during solid phase synthesis

Conjugation Examples
To demonstrate the versatility and breadth of utility of Solulinks HyNic/4FB conjugation couple, we show examples of the preparation of protein-protein, peptide-oligonucleotide, and protein-oligonucleotide conjugates (Figures 4, 5, 6). More detailed descriptions can be found in the Solulink white papers. Protein-Protein Conjugation Solulink has taken advantage of the efficiency of conjugation of two proteins using the HyNic/4FB couple in the presence of aniline to develop an All-in-One Conjugation kit that allows preparation of IgG-HRP, IgG-AlkPhos, and IgG-R-PE conjugates without the need for chromatographic purification. Figure 4 presents gel results of the conjugation of HyNic-IgG to 4FB-HRP. Note the near complete conversion of IgG to conjugate (lane 4). A simple centrifugation step removes excess enzyme, and yields purified conjugate.

Figure 4. Protein-protein conjugation. Coomassie blue stained gel of the conjugation of HyNic-antibody to 4FB-HRP showing protein ladder (Lane 1), HyNic-IgG (Lane 2), crude conjugation reaction (Lane 4) and purified IgG-HRP conjugate (Lane 5). It is to be that a polymeric IgG-HRP as produced here is the desired construct fro thse conjugates for optimal sensitivity.

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Peptide-Oligonucleotide Conjugation The preparation of these conjugates using maleimido/thiol chemistry is difficult to perform, difficult to reproduce, and is low yielding. The HyNic/4FB couple greatly simplifies the process as both linkers can be incorporated onto their respective sequences during their solid phase syntheses. For example, the HyNic peptide reagents can be incorporated on the N- or C-terminus, or internally during peptide solid phase synthesis (Figure 6-1, 6-2, and 6-3). The 4FB group can be incorporated on the 5- or 3-terminus of an oligonucleotide by a simple, very high-yielding conversion of a 3- or 5-amino oligo using sulfo-S-4FB. Alternatively, a 4FB-phosphoramidite for direct incorporation on the 5-terminus can be used. Simple mixing and incubation of the 4FB- oligonucleotide with a 23 fold excess of the HyNic-peptide, followed by purification by diafiltration, produces the conjugate in near quantitative yield. Protein-Oligonucleotide Conjugation The ability to efficiently and reproducibly prepare antibodyoligonucleotide conjugates has limited their exploitation in multiplexed diagnostic asssays. Solulinks HyNic/4FB bioconjugation couple, as applied to the conjugation of oligonucleotides with antibodies, is stoichiometrically efficient and high yielding, converting >95% antibody to antibody-oligonucleotide conjugate (Figure 7). Furthermore, conjugations of oligomers of 2060 nucleotides are conjugated with equal efficiency. The method is extremely mild,
Figure 6. Structures of Boc-HNA (1) and Fmoc-Lys-e-(Boc-HNA). These reagents are used to incorporate HyNic groups on the N- and C-termini of peptides, respectively. and 4FB-phosphoramidite is used to incorporate 4FB groups on the 5-terminus of oligonucleotides. Figure 5. Peptide-oligonucleotide conjugation. SDS-PAGE gel of conjugates visualized with UV back-shadow. Conjugation of two N-terminal HyNicmodified peptides 121 and 122 (Lanes 2 and 3) reacted with a 5-4FB modified 19-mer oligo (Lane 1) to yield the peptide-oligo conjugates (Lanes 4 and 5). As seen in lanes 4 and 5, conjugate formation was efficient at a 1:3 reaction stoichiometry (oligo:peptide). Conjugate bond stability was tested at 95OC in PBS for two hr (Lanes 6 and 7).

as no metals, reductants, or oxidants are used in the conjugation step. Further enhancing the efficiency of conjugation is the use of aniline as a reaction catalyst. In a standard conjugation

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Solulink Bioconjugation PrimerJanuary 2013

protocol, 5 equivalents of 4FB-oligonucleoitde are used, resulting in the conjugation of 23 oligonucleotides per antibody.

Traceable Biotin and Digoxigenin Labeling of Biomolecules with ChromaLink Linkers Accurate and controlled incorporation of haptens such as biotin

In a second breakthrough for antibody-oligonucleotide conjugation, we developed a method to purify the conjugate by adsorption to a proprietary magnetic affinity matrix that allows removal of excess 4FB-oligonucleoitde, followed by elution of the purified conjugate using mild elution buffers. The overall yield of the antibody-oligonucleotide conjugate is 3050% based on antibody recovery. The conjugate is >95% free from unconjugated HyNic-antibody and 4FBoligonucleotide. Multiple conjugates can be prepared simultaneously, satisfying the requirement for the use of this protocol to prepare multiple antibody-oligonucleotide conjugates for highly multiplexed detection of antigens.

and digoxigenin on biomolecules continues to be problematic due to the inherent lack of an internal chromophore on these tags. To overcome this problem, Solulink incorporated its UV-traceable (354 nm) bis-arylhydrazone chromophore into the linker arm in both biotin and digoxigenin modification reagents, the ChromaLink Biotin and ChromaLink Digoxigenin (Figure 8). Spectrophotometric quantification of the incorporation of these chromophoric haptens is straightforward and highly reproducible. Simply measuring the A280 and A354 absorbance of the modified proteins yields both the protein concentration and number of incorporated labels (Figure 7).

Figure 7. Antibody-oligonucleotide conjugation. The silver stained SDS-PAGE gel presents data for the conjugation and purification of a 40-mer (Lanes 2 and 3) and a 20-mer (Lanes 6 and 7) 4FB-oligonucleotides to HyNic-modified antibodies. In the case of the 40-mer oligonucleotide-antibody conjugate, it is evident that there is virtually no free antibody in the conjugate. In both purified conjugates, there is no visible free oligonucleotide. The broad conjugate bands are due to a distribution of 24 oligonucleotides conjugated to each antibody.

Figure 8. Structures of ChromaLink Biotin and ChromaLink Digoxigenin reagents. UV spectra of unmodified IgG and ChromaLink Biotin-modified IgG.

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For antibody labeling, Solulink designed and produced the ChromaLink Biotin and ChromaLink Digoxigenin One-Shot Kits , which include everything required to label, purify, and quantify incorporation of these labels from a single 100 g quantity of antibody. Solulink has also developed the more flexible ChromaLink Biotin Protein Labeling Kit, used in the controlled traceable biotinylation of any protein ranging from 20 to 200 kDa in size, in concentrations ranging from 0.25 to 10 mg/mL. Figure 9 shows the use of biotin- and digoxigenin-modified antibodies in a multiplex immunohistochemistry staining experiment.

Biomolecule Immobilization Immobilization of proteins, especially antibodies, is critically important in many biological applications; including immunoprecipitation, cell enrichment, antigen purification, and immunodetection. For this reason, researchers often require that proteins be immobilized on either a magnetic or non-magnetic surface. There are two main methods used to immobilize proteins: 1) immobilization of biotinylated proteins on streptavidin-modified surfaces or 2) direct covalent immobilization on activated surfaces. Solulink has engineered its conjugation chemistry to offer both alternatives within the NanoLink and MagnaLink family of products.

Figure 9. Biotin- and digoxigenin-modified antibodies in a multiplex immunohistochemistry staining experiment. Brain tissue from rats (Sprague Dawley, 200 g) were initially incubated with unconjugated primary antibody against the first target protein followed by detection with anti-primary secondary antibody conjugated to NorthernLights NL-493 tag (green fluorescence). Subsequently, the same tissue sections were incubated with a mixture of same species biotin-labeled primary antibody (against the second target protein) and DIG-labeled primary antibody (against the third target protein) followed by detection using a mixture of Streptavidin NorthernLights NL-557 tag (red fluorescence) and anti-DIG secondary antibody conjugated to a Cy5 tag (fluorescence in the far red spectral region).

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Solulink Bioconjugation PrimerJanuary 2013

Streptavidin Immobilization NanoLink Streptavidin Magnetic Beads (0.8 m) and MagnaLink Streptavidin Magnetic Beads (2.8 m) possess 3 to 15 times greater biotin binding capacity than any other commercially available magnetic bead (Figures 10,11). Solulink accomplished this by polymerizing streptavidin on an activated bead surface using its proprietary HyNic/4FB chemistry. Higher binding capacity permits the use of less solid phase (beads) to capture a given amount of a biotinylated target protein or biomolecule, resulting in proportionally lower backgrounds and cost.
Figure 11. Comparison of free biotin binding capacity of Solulinks NanoLink and MagnaLink streptavidin-coated magnetic beads and competitor beads.

Figure 10. Comparison of magnetic beads. MagnaLink and NanoLink Streptavidin Magnetic beads and their corresponding solution phase image (light microscope 400x).

Figure 12. Scheme presenting the chemistry used to convalently immobilize HyNic-proteins on 4FB beads.

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Direct Immobilization Current methods to covalently immobilize proteins on magnetic and non-magnetic surfaces are similar in that chemically reactive surfaces such as epoxy, tosyl , or NHS functional groups are mixed with biomolecules in anticipation that lysine groups located on the protein surface will covalently bind to the activated surface. There are several problems associated with this paradigm, as immobilization which is directed to lysine groups on proteins is uncontrolled, leading to over-modification and loss of protein function. In addition, for each protein to be immobilized, pH and protein levels must be optimized on a case-by-case basis to ensure optimal functionality. . It is well known that protein immobilization efficiency using these chemistries is often low and can be accompanied by significant amounts of nonspecific binding and/or leaching. These limitations lead to suboptimal assay performance and reproducibility. Solulink has overcome many of these problems by offering an immobilization technology that is highly efficient and reproducible. Solulinks protocols differ (Figure 12) in that the protein to be immobilized is first controllably and reproducibly modified with HyNic groups, then the HyNic-modified protein (or peptide) is simply mixed with a 4FB-activated surface. In this way, protein immobilization efficiency consistently exceeds 75%, with unmatched batch-to-batch reproducibility.

Summary Solulinks HyNic/4FB-based bioconjugation technology has been engineered to efficiently prepare all types of biomolecular conjugates. In most cases, Solulinks conjugation technology is far superior to classical methods and better satisfies the stringent requirements demanded by diagnostic and therapeutic products. As a case in point, Solulinks oligonucleotide-antibody conjugation technology was used during the development of the PCR-based Proximity Ligation Assay8, 17, 20 that is recognized as the most sensitive protein detection assay.

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Solulink Bioconjugation PrimerJanuary 2013

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