The conjugation of enzymes, especially alkaline phosphatase and horseradish peroxidase, to oligonucleotides has become very significant in the production of diagnostic probe systems. These developments, along with continued interest in labelling with thiol- specific tags, have prompted us to rethink routes to thiol-modified oligonucleotides, as described.below.
Thiol-Modifiers for the production of sulfhydryl groups at the 5'-terminus have been readily available commercially, including from Glen Research. These products are basedl,2,3 on blocking the thiol group during synthesis with a trityl protecting group. Although this procedure has been used successfully, several problems exist including a low level of oxidative detritylation during oligonucleotide synthesis and the use of silver nitrate during final deblocking. A recent note4describes a procedure to modify a 5'-amino-oligonucleotide to a thiol using N-acetyl-DL-homocystein thiolactone.
Synthetic routes to oligonucleotides containin 3'-thiols have also been described5,6,7 utilizing solid supports containing disulfide linkages. 3'-Thiol-modified oligonucleotides are especially interesting in cases where a different label is desired for the 5'-terminus.
Add Thiol-Modifier C6 S-S at the 5'-terminus of the oligonucleotide in the automated DMT-off synthesis mode. The DMT release from the last cycle can be used to determine coupling efficiency. To· the standard ammonium hydroxide used for deprotection, add dithiothreitol (DTT) to a concentration of 0.05M. Carry out deprotection in the normal manner (typically 55°C/16h). This procedure removes the base protecting groups and cleaves the disulfide linkage to generate the 5'-thiol. Isolate, desalt, and, if necessary, purify the thiol-modified oligo using standard procedures.
Add Thiol-Modifier C6 S-S at the 5'-terminus of the oligonucleotide in the automated DMT-on synthesis mode. Carry out deprotection in the normal manner. Purify the trityl containing oligonucleotide by HPLC or on a Poly-Pak™ cartridge omitting the 2% TFA step. Evaporate the product solution to dryness. Cleave the disulfide linkage using DTT (0.04M in 0.17M phosphate buffer, pH8) for 16h. Desalt the oligonucleotide on a cartridge or column. (The DMT containing thiol will remain attached to the desalting matrix.)
Add Thiol-Modifier C6 S-S to any nucleoside support or use Thiol-Modifier C3 S-S CPG to synthesize the desired oligonucleotide. To the standard ammonium hydroxide used for deprotection, add dithiothreitol (DTT) to a concentration of 0.05M. Carry out deprotection at 55°C for 16h. This procedure removes the base protecting groups and cleaves the disulfide linkage to generate the 3'-thioL Isolate, desalt and, if necessary, purify the thiol-modified oligonucleotide using standard procedures.
Note: Thiol-modified oligonucleotides should be kept either under an inert atmosphere or in a solution containing DTT (0.01M) to avoid oxidative disulfide formation. If necessary, the DTT can be extracted from the solution using ethyl acetate. Continue to the conjugation reaction.
1) B.A. Connolly and R. Rider, Nucleic Acids Res., 1985, 13, 4485.
2) B.A. Connolly, Nucleic Acids Res., 1987, 15, 3131-3139 ,
3) N.D. Sinha and R.M. Cook, Nucleic Acids Res., 1988, 16, 2659.
4) A. Kumar, S. Advani, H. Dawar, and G.P. Talwar, Nucleic Acids Res., 1991, 19, 4561.
5) R. Zuckermann, D. Corey, and P. Schultz, Nucleic Acids Res., 1987, 15, 5305.
6) K.C. Gupta, P. Shanna, S. Sathyanarayana, and P. Kumar, Tetrahedron Lett., 1990, 31, 2471-2474.
7) U. Asseline, E. Bonfils, R. Kurfurst, M. Chassignol, V. Roig, and N.T. Thuong, Tetrahedron, 1992, 48, 1233-1254.