3′-Azido-2′,3′-ddCTP

Product Information

A nucleotide analog for selective 3`end labeling of nucleic acids via click chemistry or Staudinger ligation

Overview and Molecular Structure

3′-Azido-2′,3′-ddCTP is a modified nucleotide analog of cytidine triphosphate (CTP), featuring a 3`azido group at the 3′ position instead of the natural 3`OH group. As a dideoxy nucleotide, the lack of 2`OH group, making it a chain terminating nucleotide during polymerase reactions.
Unlike internal labeling nucleotides (e.g., EdUTP or EUTP) 3′-Azido-2′,3′-ddCTP specifically designed for post-synthetic 3′-end modification. It is incorporated enzymatically using terminal deoxynucleotidyl transferase (TdT) or poly(A) polymerase, ensuring single-site labeling at the 3′ end.

Chemical functionality of 3′-Azido-2′,3′-ddCTP:

The azido group is a biorthogonal functionality, meaning it does not interfere with natural biomolecules. It reacts selectively with:

• Alkynes → forming triazoles via CuAAC (copper-catalyzed) or SPAAC (copper-free, using strained alkynes like DBCO)
• Phosphines → forming amides via Staudinger ligation

Both reactions meet the criteria for click chemistry, and the development of azide–alkyne cycloaddition was recognized with the 2022 Nobel Prize in Chemistry.

Typical conjugation partners include fluorescent dyes, biotin, linkers, and advanced ligands such as sugars for targeted delivery.

Application areas for 3′-Azido-2′,3′-ddCTP

• 3′-End Labeling of Nucleic Acids: Incorporation into ssDNA or mRNA followed by click conjugation for imaging or functionalization.
• Reverse Transcription and cDNA Synthesis: Acts as a chain terminator during reverse transcription, enabling controlled fragment generation for sequencing workflows.
• Next-Generation Sequencing (NGS): Used in ClickSeq kits as a chain terminator, 3′-Azido-2′,3′-ddCTP generates azido-modified fragments that enable primer or adapter conjugation via click chemistry, eliminating the need for enzymatic ligation. Like Sanger sequencing, which relies on ddNTPs to stop elongation for sequence determination, ClickSeq applies the same principle using azido-modified dideoxynucleotides. These nucleotides terminate synthesis at defined positions and introduce an azide group, allowing click chemistry-based adapter or primer conjugation without enzymatic ligation. This approach combines the precision of Sanger termination with the flexibility of modern biorthogonal chemistry, reducing artifacts such as chimeras, recombination, and ligation bias, while preserving library complexity and improving reproducibility in NGS workflows.
• Advanced Bioconjugation: Enables attachment of targeting moieties for active transport into cells.

Advantages

• Site-specific labeling at the 3′end
• Guaranteed single incorporation due to chain termination
• Compatible with copper-free click chemistry for sensitive biomolecules
• Ideal for sequencing and controlled fragment generation.

LITERATURE

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry, M. L. Winz et al., 2012, Nucleic Acids Res., Vol. 40, p. 1–13.

https://doi.org/10.1093/nar/gks062

Chemoenzymatic Preparation of Functional Click-Labeled Messenger RNA, S. Croce et al., 2020, ChemBioChem, Vol. 21, p. 1641-1646.

https://doi.org/10.1002/cbic.201900718

Application and design considerations for 3′-end sequencing using click-chemistry, M. K. Jensen et al., 2021, Methods in Enzymology, Vol. 655, p. 1-23.

https://doi.org/10.1016/bs.mie.2021.03.012

Trimannose-coupled antimiR-21 for macrophage-targeted inhalation treatment of acute inflammatory lung damage, C. Beck et al., 2023, Nature Communications, Vol. 14, 4564.

https://www.nature.com/articles/s41467-023-40185-1