Nonenzymatic oligomerization of activated nucleotides on hairpin templates.

This unit describes a protocol for nonenzymatic oligomerization of activated ribonucleotides on DNA hairpins appended by templates containing threofuranosyl nucleic acid (TNA). TNA-cytidylate templates effectively promote oligomerization of 2-MeImpG, and give 3',5'-linked oligomerization products predominantly, with good base-pairing fidelity. Although the rates of oligomerization depend on TNA content, after 3 days of incubation, oligomerization products are apparent, and full-length products are present after 10 days. Characterization of product phosphodiester bond regiochemistry is accomplished by digestion with RNase T1. Additionally, exposure of oligomerization products to calf intestinal alkaline phosphatase enables detection of any endcapping due to pyrophosphate formation. Base-pairing fidelity is assessed by challenging the template to oligomerize 2-MeImpA. The protocols described for nonenzymatic, template-directed synthesis in this unit are applicable to oligomerization of activated monomers on templates of different compositions, with respect to both base identity and polymer backbone.