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   Efficient Fluorescent DNA Labeling using Copper-free Click-Chemistry
   
       
       
  
   
   

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Click Chemistry has become a powerful method for DNA modification. Once an alkyne or azide moiety is incorporated into DNA, it can subsequently be conjugated by a simple CLICK reaction[1] with a reporter molecule such as biotin or a fluorescent dye.

A very prominent version of CLICK is the so-called "Cu(I)-free [2+3] cycloaddition strategy" that is based on strained cyclooctynes. Their use decreases the activation energy for the CLICK reaction, enabling it to be carried out at low temperatures, without the need for catalysis, and with greater efficiency than conventional CLICK[2-7].

We report here a universal DNA labeling strategy that relies on efficient PCR-incorporation of the CLICKable dUTP analogs Azide-PEG4-aminoallyl-dUTP (Figure 1; JBS Cat.No.: NU-1705), and 5-DBCO-dUTP (Figure 2; JBS Cat.No.: CLK-T09) into a DNA fragment.

Figure 1:
Chemical structure of the CLICKable dUTP analog Azide-PEG4-aminoallyl-dUTP (JBS Cat.No.: NU-1705)

Figure 2:
Chemical structure of the CLICKable dUTP analog 5-DBCO-dUTP (JBS Cat.No.: CLK-T09)



   
   Azide-PEG4-aminoallyl-dUTP
   

PCR with Taq polymerase and substitution of up to 80 % of TTP with Azide-PEG4-aminoallyl-dUTP in the dNTP mix proceeds with high yields (Figure 3). After purification, the PCR product containing the azide is efficiently coupled with Dibenzylcyclooctyne-Fluor 488 (DBCO-Fluor 488, JBS Cat.No.: CLK-A106) in a Copper-free CLICK reaction. Optimal labeling performance (approx. 5 %) is achieved with 40-80 % replacement of TTP with Azide-PEG4-aminoallyl-dUTP (Figure 4).

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Figure 3:
PCR on a 500 bp DNA with Azide-PEG4-aminoallyl-dUTP instead of TTP (1,8 % agarose without EtBr); Lane 1: 200 bp ladder, Lanes 2-7: 0 %, 20 %, 40 %, 60 %, 80 %, and 100 % replacement of TTP with Azide-PEG4-aminoallyl-dUTP, respectively.

Figure 4:
Dependence of DNA yield and labeling rate on the degree of TTP substitution. A 40-80 % replacement of TTP with Azide-PEG4-aminoallyl-dUTP is recommended.




   
   5-DBCO-dUTP
   

5-DBCO-dUTP can be incorporated by PCR into a DNA fragment with Taq polymerase with a substitution of up to 80 % of TTP in the dNTP mix (Figure 5). After purification, the PCR product containing the strained cyclooctyne moiety was efficiently coupled with Fluorescein-Azide (6-FAM Azide, JBS Cat.No.: CLK-FA001) in a Copper-free CLICK reaction (Figure 6). DNA yields after PCR are somewhat lower compared to Azide-PEG4-aminoallyl-dUTP, but higher dye labeling rates of up to 9 % are obtained.

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Figure 5:
PCR on a 500 bp DNA with 5-DBCO-dUTP instead of TTP (1,8 % agarose without EtBr); Lane 1-6: 0 %, 20 %, 40 %, 60 %, 80 %, and 100 % replacement of TTP with 5-DBCO-dUTP, respectively. Lane 7: 200 bp ladder

Figure 6:
Dependence of DNA yield and labeling rate on the degree of TTP substitution. A 40-50 % replacement of TTP with 5-DBCO-dUTP is recommended.

In conclusion, both nucleotide analogs Azide-PEG4-aminoallyl-dUTP and 5-DBCO-dUTP are well suited for PCR-incorporation of reactive groups into DNA and subsequent selective labeling.


For our entire portfolio of Click Chemistry Reagents please refer to:

www.jenabioscience.com/cms/en/1/browse/1379/


Selected references:
[1] El-Sagheer et al. (2010) Click Chemistry with DNA. Chem. Soc. Rev. 39:1388.
[2] Debets et al. (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cyclo-addition. Chem. Commun. 46:97.
[3] Bertozzi et al. (2010) Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones. J. Am. Chem. Soc. 132:3688.
[4] Bertozzi et al. (2009) Biorthogonal Chemistry: Fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48:6974.
[5] Ning et al. (2008) Visualizing Metabolically Labeled Glycoconjugates of Living Cells by Copper-Free and Fast Huisgen Cycloadditions. Angew. Chem. Int. Ed. 47:2253.
[6] Bertozzi et al. (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. U.S.A. 104:16793.
[7] Bertozzi et al. (2006) A comparative study of bioorthogonal reactions with azides. Chem. Biol. 1:644.


Nucleotide analogs not on stock may be available as custom synthesis, just send us an e-mail at nucleotides@jenabioscience.com with any questions you may have!




   
   

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