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.

An artificial gene comprising 183 base pairs has been assembled by template-directed condensation of 35- to 53-membered oligodeoxyribo nucleotides with cyanogen bromide as a condensing agent. The reaction is complete within several minutes at 0 degrees C in buffer. The resulting mini-gene was cloned and expressed in vivo and in vitro. We have also found that the polymerase inhibition technique (toe-printing) is a good way to ascertain that translation initiation complexes form in the case of single-stranded DNAs as well. Thus, along with the fully chemical assembly of synthetic genes, a rapid and sufficiently reliable method for determining their ribosome-binding properties was developed.

The chemical ligation of 17 50-54-membered nicked DNA dumbbells with different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on the sequence of the closing fragments, nick position, and nucleotides facing the nick. Coupling yields of 80%-90% were observed when the nick was situated in the middle of the molecule between two T residues or was adjacent to GCGA2GC or GCGA3GC minihairpins. In the last case, the reacting 3'-phosphate and 5'-hydroxy groups were brought close together by only two base pair minihairpins. The coupling yields did not depend on the nature of the condensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical synthesis of closed dumbbells.

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