Control of reaction chemoselectivity with a circular DNA template in the ligation of short oligodeoxyribonucleotides.

A study has been conducted to examine the chemoselective control attained in the ligation of short oligodeoxyribonucleotides (ODNs) directed by association on a circular DNA template through triplex formation relative to the same ligation on a single-strand DNA template through duplex formation. The highly efficient, chemospecific ligation achieved under a variety of conditions on the circular DNA template presumably arises from the higher substrate-template association resulting in improved transition state accessibility and protection of the ODNs from side reactions.

Chemical ligation of oligodeoxyribonucleotides on circular DNA templates.

We report the use of small circular DNA as a triplex-directing template for the highly efficient chemical ligation of oligodeoxyribonucleotides (ODNs) using cyanogen bromide (BrCN). These investigations compared the use of a linear homopyrimidine DNA template (17mer) and a circular pyrimidine-rich DNA template (44mer) for directing the chemical ligation of two homopurine ODNs (6mer + 11mer). The effects of substrate/template ratio, buffer, salt, ionic strength, pH and temperature have been examined in the BrCN activated ligation reactions. The optimal yield of 51% for ligation on the linear template was at pH 6.0, 200 mM MgCl2, 4 degreesC. In contrast, near quantitative ligation on the circular template occurred at higher pH, higher temperature, and showed less dependence on Mg2+concentration (97% yield, pH 7.5, 200 mM MgCl2, 25 degreesC). The relative observed rate of the ligation reaction was a minimum of 35 times faster on the circular DNA template relative to the linear template at pH 7.5, 200 mM MgCl2, 4 degreesC. These investigations reveal that chemical ligation of short ODNs on circularized DNA templates through triplex formation is a highly efficient process over a broad range of conditions.