Product Information

A biotinylation reagent for labeling of azido modified targets by strain-promoted azide alkyne cycloaddition (SPAAC)

What is biotin

Biotin, also known as vitamin B7, is a heterocycle which plays an important role in biological systems. It functions as a coenzyme in carboxylation reactions, for example in fatty acid biosynthesis. In addition to its biological importance, biotin is known for forming one of the strongest non-covalent interactions with the proteins avidin and streptavidin. This exceptionally stable interaction is widely exploited in modern biochemical research, particularly in peptide, protein, and nucleic acid applications.

Applications of biotin

Peptide/protein research:

The biotin–streptavidin (or avidin) interaction enables a wide range of applications in peptide and protein research. Biotinylation is commonly used for affinity purification, where biotin labeled target proteins or protein complexes bind to streptavidin or avidin coated magnetic beads or resins, allowing rapid one step purification.

Biotin labeling is also extensively applied in detection assays through the use of streptavidin conjugated to reporter molecules, most commonly fluorophores or enzymes. This approach is widely used in immuno-assays such as ELISA, Western blotting, and immunohistochemistry. In addition, biotinylation facilitates the enrichment of specific protein populations, for example newly synthesized proteins, for downstream mass spectrometry analysis.

Nucleic acid research:

In nucleic acid research biotin labeling is mostly used for purification processes. Again, the biotin bonding to Streptavidin/Avidin containing beads or resins is utilized to bind the nucleic acids and separate them from impurities. Biotin containing probes can be used for in situ hybridization (ISH) to bind to specific DNA/RNA structures. The probes can be visualized and localized afterwards by binding to Streptavidin/Avidin based markers.

Biotin Labeling via Click Chemistry

All these applications require reliable access to biotin labeled biomolecules. Click chemistry provides a simple, cost effective, and highly selective approach to biotinylation. For click based labeling, the biomolecule of interest must carry either an azide or an alkyne functionality.

Azide or alkyne modified biomolecules, as well as biotinylation reagents such as DBCO-PEG4-Biotin, are commercially available. baseclick offers alkyne or azide modified oligonucleotides and mRNA, as well as pre biotinylated nucleic acids. For other targets or custom applications, feasibility can be discussed via support@baseclick.eu.

Chemical Properties of DBCO-PEG4-Biotin

DBCO-PEG4-Biotin is a highly versatile biotinylation reagent designed for labeling under very mild conditions. Conjugation is achieved via strain promoted azide–alkyne cycloaddition (SPAAC), a copper free click reaction that requires neither heating nor a catalyst, in contrast to CuAAC. The reaction forms a stable triazole linkage with azide modified targets, making it particularly suitable for in vivo labeling applications or other copper sensitive systems.

Azide–alkyne cycloaddition reactions are considered bioorthogonal, as they do not interfere with native biological functionalities. DBCO-PEG4-Biotin further incorporates a hydrophilic PEG4 spacer, which reduces steric hindrance between biotin and the labeled biomolecule while increasing overall water solubility.

These chemical features make DBCO-PEG4-Biotin an ideal choice for biotin labeling of azide modified biomolecules, including peptides, proteins, and nucleic acids, especially when the use of copper must be avoided.

Because click chemistry relies on non-naturally occurring functional groups, the site of biotin attachment can be precisely controlled, in contrast to traditional biotinylation methods such as NHS ester-based chemistry.

LITERATURE

DNA Adduct Detection after Post‐Labeling Technique with PCR Amplification (DNA‐ADAPT–qPCR) Identifies the Pre‐ribosomal RNA Gene as a Direct Target of Platinum–Acridine Anticancer Agents, X. Yao et al., 2021, Chemistry–A European Journal, Vol. 27(59), p. 14681-14689.

https://doi.org/10.1002/chem.202102263

Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch, Y. Wang et al., 2018, Chemical Science, Vol. 10(2), p. 447-452.

https://doi.org/10.1039/C8SC04272A

O-GlcNAcylation of blimp-1 in lymphocytes inhibits its transcriptional function and is associated with migration and invasion of breast cancer cells, Y. F. Chen et al., 2022, Molecular Cancer Research, Vol. 20(4), p. 650-660.

https://doi.org/10.1158/1541-7786.MCR-21-0405