3′-Azido-2′,3′-ddTTP

Product Information

A Nucleotide Analogue for the Selective 3´End Labeling of Nucleic Acids by Click Chemistry (CuAAC/SPAAC) or Staudinger Ligation

3′-Azido-2′,3′-ddTTP is a chemically modified analogue of Uridine triphosphate (UTP) featuring an azido (N₃) group replacing the 3′-OH group of the sugar moiety. This modification enables efficient labeling through click chemistry reactions:

• CuAAC (Copper-catalyzed alkyne–azide cycloaddition) – Produces a single regioisomer and is easily scalable for product development.
• SPAAC (Strain-promoted alkyne–azide cycloaddition) – Catalyst-free, simplifying purification, but yields two regioisomers.

The absence of the 3′-OH group makes 3′-Azido-2′,3′-ddTTP a chain-terminating nucleotide in polymerization reactions such as IVT. Unlike other modified DNA or RNA nucleotides (e.g., EdUTP or EUTP), it cannot be incorporated internally during nucleic acid synthesis. Instead, it is ideal for post-synthetic, 3′-end-specific introduction of a single azido-modified nucleobase using enzymes such as T7 polymerase or terminal deoxynucleotidyl transferase (TdT).

This nucleotide can replace T in DNA or U in RNA for 3′-end labeling. Detailed protocols for conjugation with fluorescent dyes, biotins, linkers, and cell-targeting ligands via click chemistry are available from baseclick.

Role in NGS Sequencing

The chain-terminating property of 3′-Azido-2′,3′-ddTTP is utilized in baseclick’s ClickSeq NGS kits for generating cDNA libraries, improving sequencing workflows by reducing artifacts and enhancing reproducibility.

Azide Functionality and Bioorthogonal Ligation

The azido group (R–N₃) is bioorthogonal, meaning it does not react with natural biomolecules. Under physiological conditions, azides selectively react with:

• Alkynes → Form triazoles via Cu(I)-catalyzed or strain-promoted cycloaddition (e.g., DBCO, BCN).
• Phosphines → Form amides via Staudinger ligation.

These reactions enable precise chemical modifications without interfering with biological systems.

Application areas

• 3′-End labeling of nucleic acids (ssDNA, mRNA) for imaging, affinity tagging, or functionalization via click chemistry.
• Sequencing workflows:
  • Acts as a chain-terminating nucleotide during reverse transcription or cDNA synthesis.
  • Enables primer conjugation without enzymatic ligation, improving efficiency and reducing artifacts in RNA-seq workflows.

Used in baseclick and ClickSeq kits to overcome common RNA-seq bottlenecks such as chimeras, recombination, fragmentation bias, and ligation inefficiencies, while preserving library complexity.

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