Oligonucleotide circularization by template-directed chemical ligation

An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpin-like structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.