What are Biotinylation and Biotinylated PEG?

Biotinylation is the process of covalently attaching biotin to molecules (such as amino acids, peptides, antibodies, or proteins). Due to the small molecular weight of biotin, biotinylation is usually rapid and specific and is unlikely to interfere with the inherent function of the molecule. Biotin has extremely high affinity, rapidity and high specificity with streptavidin or avidin. Therefore, biotinylation is widely used in biomedical fields, and biotinylated proteins are widely used in biotechnology as a molecular tool.

What is Biotin?

Biotin, a B vitamin, is an essential nutrient found naturally in certain foods and is available as a dietary supplement. Biotin is also known as vitamin H, D-biotin, coenzyme R or W factor. This water-soluble vitamin is a component of five carboxylase enzymes (propionyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase [MCC], acetyl-CoA carboxylase 1, and acetyl-CoA carboxylase Cofactor for carboxylase 2), which catalyze key steps in fatty acid, glucose, and amino acid metabolism. Biotin also plays a key role in histone modification, gene regulation (by altering the activity of transcription factors), and cell signaling.

Fig. 1. Structure of biotin.

From a biotechnological perspective, the most attractive property of biotin is its unusually high affinity and highly specific binding to avidin and streptavidin of bacterial origin. In fact, the binding of biotin and avidin is one of the strongest non-covalent interactions known, with dissociation constants in the range of 10^-14 M to 10^-15 M. This super-strong interaction makes biotin highly tolerant to a variety of harsh experimental conditions, such as extreme pH, temperature, and buffer conditions. Therefore, biotin is widely used in biomedical applications for the detection, immunoprecipitation, or purification of molecules of interest.

What is the Biotin-Avidin System?

Biotin-Avidin-System (BAS) is a new type of biological reaction amplification system developed in the late 1970s. The high-affinity strong binding between biotin and avidin and the multi-level amplification effect make BAS immunolabeling and related tracer analysis more sensitive. With the deepening of the research on the three-dimensional structure, binding characteristics and stability of biotin and avidin, the application of BAS and its related technologies in a variety of automated immunoassay techniques has further promoted the development of sensitive, rapid and accurate determination techniques for some trace substances in plasma and body fluids such as small molecule polypeptide hormones, tumor markers, myocardial injury markers and blood drug concentration analysis. It also shows obvious advantages in nucleic acid probe labeling, separation and purification of cells and biologically active substances.

Biotin-labeled Proteins and Peptides

The valerate side chain of the biotin molecule can be derivatized to incorporate a variety of reactive groups that are used to link biotin to other molecules. Using these reactive groups, biotin can be easily attached to most proteins and peptides. Biotinylation reagents can be used to target a variety of functional groups, including primary amines, thiols, carbohydrates, and carboxyl groups. There are also options for photosensitive biotin compounds that respond nonspecifically to photoactivation. This functional group-specific variety is useful in selecting biotinylation reagents that do not inactivate the target macromolecule.

Fig. 2. Biotinylated Protein and DNA (ACS Chem. Biol. 2022, 17(12): 3270-3275).

The solubility of biotinylation reagents will largely affect the labeling ability of proteins or other macromolecules. Proteins have hydrophilic and hydrophobic regions depending on the side chains of the amino acids and the protein configuration, and these regions can promote or hinder the biotinylation process depending on the solubility of the biotinylation reagent. Some studies require biotinylated proteins that are highly hydrophilic. For example, when studying the expression of molecules on the surface of cells or the internalization process of tracer surface molecules, it is necessary for the biochemical protein to be hydrophilic to limit the biotinylated-labeled protein from passing through the hydrophobic cell membrane and thus remaining on the surface of the cell. Biotin with PEG chains has higher solubility, which can improve the biotinylation process of proteins and prevent the aggregation of biotinylated proteins.

Biotin-labeled Antibodies

Generally, each antibody can be labeled with 3 to 5 biotins. When labeling, the ratio of biotin to antibody is affected by the antibody concentration. When labeling with biotin, the antibody must not contain sodium azide, BSA, glycine, Tris or any other additives with free amino groups. Antibodies are usually pre-treated before biotin labeling. The more commonly used methods are: ultrafiltration with 0.1 M PBS buffer, or dialysis. Since there are many types of biotins and they are usually in the form of powder, a suitable solvent should be selected when dissolving. Biotin labeling is relatively simple. You only need to mix the processed antibody and biotin at a molar ratio of 1:10~20 (the actual ratio used should be adjusted according to the specific situation), and react at room temperature for 1 hour in the dark. After the reaction, free biotin and other reagents are removed by dialysis or ultrafiltration. Then save the biotinylated antibody in a suitable storage solution.

Fig. 3. Biotin molecule with PEG chains.

Biotin PEG

Biotin PEG is a biotin molecule attached to a PEG chain. Monodisperse PEG has well-defined chain lengths, allowing the design and study of specific biotin complexes. Due to its unique properties, biotin PEG is commonly used in a variety of biomedical and biotechnological applications. Biotin PEG can be used as a targeting moiety in drug delivery systems. By conjugating biotin PEG to a drug or drug carrier, specific targeting of cells overexpressing biotin receptors, such as cancer cells, can be facilitated. This targeted drug delivery approach can enhance therapeutic efficacy and minimize off-target effects. Furthermore, biotin PEG can be used as a probe in various diagnostic assays, such as enzyme-linked immunosorbent assay (ELISA) or lateral flow assays. Biotinylated probes can be combined with streptavidin or avidin conjugated to the detection system, allowing sensitive and specific detection of target analytes. Biotin PEG can also be incorporated into cell culture media or hydrogels to provide a functionalized surface or scaffold for cell attachment and growth. This is useful for tissue engineering applications or studying cell behavior and signaling pathways.

Reference

1. Seiple, I.B. et al. Biotin as a Reactive Handle to Selectively Label Proteins and DNA with Small Molecules. ACS Chem. Biol. 2022, 17(12): 3270-3275.