7. Click Chemistry in DNA Synthesis

a) Applications and Procedures in DNA Synthesis

Due to its unique bioorthogonality the Click reaction is very useful also in any oligonucleotide synthesis. Simple or no workup and purification of the product are further advantages. However, the use of this method for DNA modification has been somewhat delayed by the fact that copper ions damage DNA, typically causing strand breaks [1]. As these problems have now been overcome by the use of copper(I)-stabilizing ligands (e.g. tris(benzyltriazolylmethyl)amine, TBTA [2]), Carell et al. and Seela et al. discovered that the CuAAC reaction can be used to functionalize alkyne-modified DNA nucleobases with extremely high efficiency [3-5].

A broad field of applications opens by using DNA Click components:

• DNA and RNA labelling: incorporation of alkyne-phosphoramidites followed by labelling with azido markers [8];
• PCR assays, PCR primers and labelling of large fragments with alkyne triphosphates in nucleotide mixtures; labelling PCR-fragments with azido markers [8];
• oligonucleotides as multi-labelled primers for pre- or post-synthetic modification;
• FISH probes and FISH experiments: with alkyne-modified oligonucleotides and labelling with azido marker (pre- or post-hybridization);
• PEGylation of DNA and RNA;
• Microarrays with phosphoramidites, triphosphates or oligonucleotides to set up microarrays;
• Nanoparticles, Bioconjugation.

References

• [1] Oxidative Nucleobase Modifications Leading to Strand Scission; C. J. Burrows and J. G. Muller; Chem Rev 1998; 98: 1109-1152.
• [2] Polytriazoles as copper(I)-stabilizing ligands in catalysis; T. R. Chan, R. Hilgraf, K. B. Sharpless and V. V. Fokin; Org Lett 2004; 6: 2853-5.
• [3] Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA; J. Gierlich, G. A. Burley, P. M. Gramlich, D. M. Hammond and T. Carell; Org Lett 2006; 8: 3639-42.
• [4] DNA containing side chains with terminal triple bonds: Base-pair stability and functionalization of alkynylated pyrimidines and 7-deazapurines; F. Seela and V. R. Sirivolu; Chem Biodivers 2006; 3: 509-14.
• [5] Click-click-click: single to triple modification of DNA; P. M. Gramlich, S. Warncke, J. Gierlich and T. Carell; Angew Chem Int Ed Engl 2008; 47: 3442-4.
• [6] New labelling strategies for the sensitive detection of analytes; Patent WO2006/117161
• [7] Nucleosides and Oligonucleotides with Diynyl Side Chains: Base Pairing and Functionalization of 2‘-Deoxyuridine Derivatives by the CuI-Catalyzed Alkyne-Azide “Click’’ Cycloaddition; F. Seela, V. R. Sirivolu, Helv. Chim. Acta, 2007: 90, 535.
• [8] Synthesis of highly modified DNA by a combination of PCR with alkyne-bearing triphosphates and click chemistry; J. Gierlich, K. Gutsmiedl, P. M. Gramlich, A. Schmidt, G. A. Burley and T. Carell; Chemistry 2007; 13: 9486-94.

Published Examples of Click Reactions in DNA Synthesis:

These protocols may be used as a starting point with further optimization.

Preparation of the “Click Solution”:

• The “click solution” (0.1 M CuBr / 0.1 M TBTA 1:2 in DMSO/t-BuOH 3:1) must always be freshly prepared prior to use!
• Dissolve 1 mg CuBr in 70 μl DMSO/t-BuOH 3:1 to obtain a 0.1 M solution. This solution must be freshly prepared and cannot be stored.
• Dissolve 54 mg TBTA in 1 ml DMSO/t-BuOH 3:1 for a 0.1 M solution. This solution can be stored at -20 °C.
• Add 1 volume of the 0.1 M CuBr solution quickly to 2 volumes of the 0.1 M TBTA solution to obtain “click solution”, ready to use.

Click Procedure for Short DNA:

Procedure using CuBr: To 5 μl of a 2 mM DNA solution (10 nmol) in water, 5 μl of an azide solution (10 mM, 50 nmol, 5 eq.), 3 μl of a freshly prepared solution containing 0.1 M CuBr and 0.1 M TBTA ligand in a 1:2 ratio in 3:1 DMSO/t-BuOH is added. The mixture is thoroughly mixed and shaken at 25 °C for 3-4 h. The reaction is subsequently diluted with 0.3 M NaOAc (100 μl) and the DNA is precipitated using 1 ml cold EtOH. The supernatant is then removed and the residue is washed twice with 1 ml cold EtOH. The washed residue is redissolved in pure water (20 μl) and can be used without further purification.

Considerations for the CuBr method:

The labelling reaction works more efficiently with concentrated solutions of alkynes (oligo) and azides (label). The best way to carry out the click reaction is to mix the oligo and the azide-label in a minimal amount of solvent (Alkyne / Azide ratio: from 1:2 to 1:10 for high density labelling reactions, e.g. 10 alkynes in a row). The click reaction is normally accelerated by elevated temperature and can be ready in less than 30 min when the reaction temperature is around 40 - 45 °C. The reaction time depends on:

a) concentration of azide and oligo in the solution;

b) reaction temperature;

c) stirring and/or mixing of the solution.

The work-up of the reaction is normally carried out by precipitation of the labelled oligo.

Click Procedure Using Alternative Cu(I) Sources:

Procedure using TCEP: To 25 μl of a 0.5 mM DNA solution (12.5 nmol) in water, 6.25 μl of an azide solution (0.1 N, 625 nmol) and 10 μl of a solution containing Cu(II)-salt (CuSO4) and TBTA ligand in a 1:2 ratio in 4:3:1 water/DMSO/t-BuOH is added (0.05 N, 250 nmol). The mixture is vortexed and 5 μl of a freshly prepared tris-(2-carboxyethyl)-phosphine (TCEP) solution in water is added (0.1 N, 500 nmol). The solution is shaken at 15 °C over night and subsequently diluted with water (200 μl) and used for gel electrophoresis without further purification. Instead of TCEP, also sodium ascorbate can be used.

Click Procedure for a 300 bp PCR Product:

To 10 μl DNA solution (1-4 pmol DNA, 10 mM Tris), 10 μl fluorescein azide solution (5 mM, diluted with 10 mM Tris with 5 % t-BuOH from a stock of 0.1 N in DMSO) and 10 μl precomplexed Cu(I) is added (10 mM; 1 mg CuBr (99.99%) dissolved in 700 μl of 10 mM TBTA ligand in t-BuOH/DMSO 1:3). The sample is shaken at 37 °C for 2 h. Then formamide buffer is added and the samples are analyzed using a 5 % PAGE gel. Control experiments show that the reaction is completed in less than 30 min.

Additional References related to Click Chemistry and DNA Synthesis:

• A Programmable DNA-Based Molecular Valve for Colloidal Mesoporous Silica. A. Schlossbauer, S. Warncke, P. M. E. Gramlich, J. Kecht, A. Manetto, T. Carell, T. Bein, Angew. Chem. Int. Ed. 2010; 49: 4734-4737.
• Click Chemistry with DNA. A. H. El-Sagheer, T. Brown, Chem. Soc. Rev. 2010; 39: 1388-1405.
• Click Chemistry as a Reliable Method for the High-Density Postsynthetic Functionalization of Alkyne-Modified DNA. J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T. Carell, Org. Lett. 2006; 8: 3639–3642.
• Direct DNA Metallization. G. A. Burley, J. Gierlich, M. R. Mofid, S. T. H. Nir, Y. Eichen, T. Carell, J. Am. Chem. Soc. 2006; 128: 1398-1399.
• DNA Containing Side Chains with Terminal Triple Bonds: Base Pair Stability and Functionalization of Alkynylated Pyrimidines and 7-Deazapurines. F. Seela, V. Ramana Sirivolu, Chemistry & Biodiversity 2006; 3: 509.
• Formation of bimetallic Ag-Au nanowires by metallization of artificial DNA duplexes. M. Fischler, U. Simon, H. Nir, Y. Eichen, G. A. Burley, J. Gierlich, P. M. E. Gramlich, T. Carell, Small 2007; 3: 1049-55.
• Nucleosides and Oligonucleotides with Diynyl Side Chains: The Huisgen-Sharpless Cycloaddition “Click reaction’’ Performed on DNA and their Constituents. F. Seela, V. R. Sirivolu, Nucleosides, Nucleotides and Nucleic Acids 2007; 26: 597.
• DNA Photography: An Ultrasensitive DNA-Detection Method Based on Photographic Techniques. D. M. Hammond, A. Manetto, J. Gierlich, V. A. Azov, P. M. E. Gramlich, G. A. Burley, M. Maul, T. Carell, Angew. Chem. Int. Ed. 2007; 46: 4184-4187.
• Pronounced Effect of DNA Hybridization on Click Reaction Efficiency. C. T. Wirges, P. M. E. Gramlich, K. Gutsmiedl, J. Gierlich, G. A. Burley, T. Carell, QSAR Comb. Sci. 2007; 26: 1159–1164.
• Postsynthetic DNA Modification through the Copper-Catalyzed Azide-Alkyne Cycloaddition Reaction. P. M. E. Gramlich, C. T. Wirges, A. Manetto, T. Carell, Angew. Chem. Int. Ed. 2008; 47: 8350-8358.
• Click Chemistry and Oligonucleotides: How a simple reaction can do so much. F. Morvan, A. Meyer, G. Pourceau, S. Vidal, Y. Chevolot, E. Souteyrand, J.-J. Vasseur Nucleic Acids Symposium Series 2008; 52: 47-48.