Both cyanogen bromide (BrCN) and 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide may be used as coupling reagents for the template-directed assembly of DNA duplexes containing the sugar-phosphate backbone modification. Both reagents show similar ligation site structure-specific trend. Practical recommendations are given for selection of the condensing reagent depending on the properties of the duplex. Based on 31P NMR spectroscopy data, a scheme is suggested for BrCN activation of the nucleotide phosphomonoester group. Using both condensing reagents, we studied the condensation of oligonucleotides containing ribo-segments (from mononucleotide residue to full sequence) on the DNA template. Efficiency of the chemical ligation of RNA oligomers was shown to be much lower than that of DNA analogues. The coupling yield depends on the position of the RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick.

Cyanogen bromide was found to condense oligodeoxyribonucleotides on a complementary template in aqueous solution. Optimum conditions for this vigorous and effective reaction were developed. CNBr proved to be useful for incorporation of phosphoramidate or pyrophosphate internucleotide bonds in DNA duplexes.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.

No abstract

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.