Solid-Supported Synthesis, Deprotection and Enzymatic Purification of Oligodeoxyribonucleotides. M.S. Urdea and T. Horn, Tetrahedron Lett., 1986, 27, 2933.
With today's automated synthesizers, oligonucleotides are rapidly and conveniently synthesized with a high degree of efficiency. Using cyanoethyl phosphoramidite chemistry, the deprotection of the synthetic sequences is also rapid and convenient. Thus, purification, if desired, remains the last labor-intensive step for which automation is currently unavailable.
In a recent publication, Urdea and Horn of Chiron Research, describe a procedure for enriching the target oligonucleotide by enzymatic degradation of the failure sequences. The authors suggest that because this procedure involves only reagent addition and wash steps, it should be amenable to automation.
The authors adjust the protection scheme (use N', N'-dibutylformamidine instead of isobutyryl to block the N6-position of dG) and the endcapping procedure (substitute levulinic anhydride for acetic anhydride) and replace the final 5'DMT group with benzoate. These adjustments make all protecting groups except the 5'-benzoate of the target sequence and the 3'-succinate linkages to CPG labile to hydrazine.
After deprotection, failure sequences are hydrolyzed on the support with spleen phosphodiesterase, the target sequence being protected by the 5'-benzoate. Standard ammonia treatment releases the fully deprotected product in the presence of failure sequences reduced to 10-mer or less, allowing simple and rapid reverse-phase cartridge purification.
This technique may require further refinement but it does exhibit considerable promise for the eventual total automation of synthesis, deprotection, and purification of DNA.
