OLIGONUCLEOTIDE CIRCULARIZATION BY TEMPLATE-DIRECTED CHEMICAL LIGATION

An efficient method for producing the covalent closure of oligonucleot ides 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 oligo mer-template length ratio was evaluated. The efficiency of circulariza tion was shown to correlate well with the secondary structure of the p recursor oligomer (as predicted by a simple computer analysis), hairpi n-like structures bearing free termini clearly favouring the circulari zation 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 e ffective. An original set of assays was developed to confirm the circu lar structure of the covalently closed oligonucleotides.