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.
Selvasekaran
J
J
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
Turnbull
K D
KD
D020029:Base Pairing; D004247:DNA / Q000737:chemistry*; D004270:DNA, Circular / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D006860:Hydrogen Bonding; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D004247:DNA