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.

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Chimeric oligo(ribo-deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGC-CAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C,, and A,, .

* Здесь и далее префикс d (дезокси) опущен, направление связи во всех дуплексах, как в дуплексе I.