In-Line Coupling of the Enzymatic Degradation of Oligonucleotides with Capillary Polymer Sieving Electrophoresis

This paper deals with how an enzymatic reaction can be integrated in a capillary electrophoretic system when the capillary is filled with a polymer solution. The difference in electrophoretic mobility between enzyme and substrate makes two zones merge inside the capillary. The feasibility of this integrated system is demonstrated by performing and following the degradation of short model homo-oligomeric deoxynucleotides (dA21 and pdA21) with phosphodiesterase I. After 3‘-exonucleolytic breakdown, oligonucleotide fragments are separated in-line in a 4% solution of a low viscosity-grade hydroxyethyl cellulose. Degradation and separation are achieved under 25 min, the number of manipulation steps is strongly reduced, and the system is fully automated. This technique opens possibilities of screening larger libraries of synthetic oligonucleotides for individual stability toward purified nucleases.