Nucleic Acid Labeling

Nucleic acid labeling is one of the essential steps for detecting and purifying nucleic acids (DNA and RNA) in molecular biology labs. Chemical methods are the most common methods for nucleic acid labeling. Labeling agents can combine with nucleic acid fragments, including biotin, digoxigenin, phosphates, and fluorophores. Nucleic acid labeling by click chemistry has been extensively studied in recent years. In the future, more methods for nucleic acid labeling using click chemistry are expected to be developed to promote the development of quantitative biology.

Click chemistry is the most promising approach to overcome these challenges, because it has the following advantages^[1]: (1) bioorthogonal reactions; (2) mild reaction conditions suitable for fragile biomolecules, cells, and tissues; (3) extremely high reaction ratio; (4) small size of the functional groups for the cross-coupling reactions; and (6) simple metabolic labeling procedures in living cells.

Applications

• DNA labeling

Scheme 1. DNA labeling by click chemistry.

Click chemistry is a novel approach to DNA labeling, which has received widespread attention. Ju and co-workers^[1] introduced an azide tag to the 5'-end of single-stranded DNA, which was then labeled with a fluorescent tag. As shown in scheme 1, the oligonucleotide 5'-amino-GTT TTC CCA GTC ACG ACG-3'DNA 1 (M13-40 universal forward sequencing primer) was reacted with succinimidyl 5-azidovalerate 12 to produce azido-labeled DNA 2. Copper free 1, 3-dipolar cycloaddition between the alkyne 13 and the azido labeled DNA 2 was carried out at 80 °C in aqueous conditions to produce a mixture of FAM-labeled DNA 3 and DNA 4. After the reaction, excess alkyne 13 was removed by size-exclusion chromatography and the resulting labeled DNA was desalted with an oligonucleotide purification cartridge and used for DNA sequencing.

• RNA labeling

Scheme 2. Schematic
diagram of EU

The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction is shown to be compatible with RNA, thus, it can be applied to RNA labeling. Salic and Jao^[2] recently described a chemical method to label RNA for detecting its synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a CuAAC click reaction with fluorescent azides, followed by microscopic imaging. Notably, labeling was effective not only in cultured cells but also in living mice.

What Can We Do?

Alfa Chemistry has a strong research foundation in the field of nucleic acid labeling by click chemistry. We have ability to help you explore the various applications of click chemistry and provide you with related click chemistry reagents, technical advice and services. If you have problems, please don't hesitate to contact us.

References

1. Seo, T.S.; et al. Click chemistry to construct fluorescent oligonucleotides for DNA sequencing. The Journal of Organic Chemistry. 2003, 68(2): 609-612.
2. Jao, C.Y.; Salic, A. Exploring RNA transcription and turnover in vivo by using click chemistry. Proceedings of the National Academy of Sciences of the United States of America. 2008, 105 (41): 15779-15784.