2′-Azido-2′-dATP

Product Information

A Nucleotide Analogue for the Selective 3´End-Labeling

Molecular Structure and Applications of 2′-Azido-2′-dATP

2′-Azido-2′-deoxyadenosine 5´-triphosphate (2´-Azido-2´-dATP) is a chemically modified analogue of adenosine triphosphate (ATP), with an azido (N₃) group replacing the 2′ hydroxyl (OH) group of the sugar backbone.

2′-Azido-2′-dATP is designed for the site-specific, post-synthetic introduction of one to five azido groups at the 3′ end of nucleic acids, using enzymes such as poly(A) polymerase or terminal deoxynucleotidyl transferase (TdT). These azido groups enable the 3′ end to be modified with fluorescent dyes, biotins, linkers, and cell-targeting ligands via click chemistry.

2′-Azido-2′-dATP is also ideal for generating internally modified RNAs. When added to an in vitro transcription (IVT) reaction mixture, RNA polymerases efficiently incorporate 2′-azido-2′-dATP into the nascent RNA strand. These RNA transcripts can then undergo further modification with high specificity and sensitivity using bioorthogonal click chemistry.

The azido group: Biorthogonal RNA Labeling via Click Chemistry

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

• alkynes, forming triazoles via copper-catalysed azide–alkyne cycloaddition (CuAAC) or copper-free strain-promoted azide–alkyne cycloaddition (SPAAC).
• phosphines, forming amides via Staudinger ligation.

Both reactions fulfill the criteria for click chemistry, enabling the highly efficient ligation of labels as fluorescent dyes, biotins, linkers, or cell-targeting ligands to nucleic acids. The development of the azide–alkyne cycloaddition was recognized with the Nobel Prize in Chemistry in 2022.

Application Areas for 2′-Azido-2′-dATP

• 3′-End Labeling of Nucleic Acids: post-transcriptional enzymatic incorporation / addition to the 3´-end, followed by click conjugation with dyes, biotin, or ligands.
• Internal Labeling of RNA: co-transcriptional incorporation into RNA by in vitro transcription (IVT), followed by click conjugation with dyes, biotin, or ligands.
• Advanced Bioconjugation: allows the attachment of sugars or targeting moieties for active mRNA transport into cells.
• Copper-Free Click Chemistry In Vivo: the azido group of 2′-Azido-2′-dATP is a biorthogonal functionality, meaning that it does not interfere with natural biomolecules.
• Compatible with Enzymatic Incorporation: 2′-azido-2′-dATP is accepted as substrate by T7 RNA polymerase, poly(A) polymerase, and TdT.

Protocols and Resources

baseclick provides detailed protocols for:

• 3′-End modification of nucleic acids using 2′-azido-2′-dATP
• Click conjugation with fluorescent dyes, biotin, linkers, and targeting ligands

LITERATURE

Bioorthogonal mRNA labeling at the poly (A) tail for imaging localization and dynamics in live zebrafish embryos, K. J. Westerich et al., 2020, Chemical Science, Vol. 11(11), p. 3089-3095.

https://doi.org/10.1039/C9SC05981D

Enzymatic Generation of Double‐Modified AdoMet Analogues and Their Application in Cascade Reactions with Different Methyltransferases, M. Erguven et al., 2022, ChemBioChem, Vol. 23(24), e202200511.

https://doi.org/10.1002/cbic.202200511

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(10), p. e78. https://doi.org/10.1093/nar/gks062

Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells. L. Anhäuser et al., 2019, Nucleic Acids Res., Vol. 47(7), p. e42. https://doi.org/10.1093/nar/gkz084

Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. C. W. Tornøe, C. Christensen, M. Meldal, 2002, J. Org. Chem., Vol. 67, p. 3057-3064. https://doi.org/10.1021/jo011148j

A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, 2002, Angew. Chemie Int. Ed., Vol. 41, p. 2596–2599. https://doi.org/10.1002/1521-3773(20020715)41:14%3C2596::AID-ANIE2596%3E3.0.CO;2-4