Figure 5.

Convergent ligase catalyzed strategies for therapeutic oligonucleotide synthesis from short fragments. (a) Assembly of fragments annealed to a complementary template using DNA ligases. Impurity fragments with incorrect sequences do not anneal to the template and are removed by filtration. Following ligation, the oligonucleotide product is melted from the template and isolated by filtration, and the template is recycled. (b) Double-stranded oligonucleotides such as siRNAs can be constructed from overlapping, complementary fragments using a DNA ligase. (c) A nontemplated approach to oligonucleotide synthesis exploiting RNA ligases. 3′-Phosphate protecting groups are required to control the order of fragment assembly. Following ligation the 3′-end is dephosphoryated using alkaline phosphatase. The dashed lines between nucleotides indicate that some bases have been omitted from the diagram.