Chemical Synthesis and Biological Applications of Nucleic Acids to Nitrogen Fixation

摘要

Progress in the synthesis of oligonucleotides using a solid phase support is reported. A bench top solvent delivery system, a shaker assembly, and reaction vessels have been constructed for conducting these synthetic operations. Succinoylated mononucleotides were prepared and coupled to the polymer support, and a cytidine derived polymer was used for construction of the hexanucleotide, d(G-G-T-A-C-C). Various analytical techniques were used to monitor the synthesis and cleavage stages. Synthesis demonstrated that the polymer support method has several advantages over traditional solid phase synthesis. Alternate methods were investigated for synthesizing methyl triesters without success. The phosphite synthetic method for construction of an oligonucleotide requires significant refinements to compete with the standard triester solution or solid phase synthetic approaches. An undecanucleotide probe for a specific nif protein sequence was prepared and labeled with phosphorus 32p. Binding assays indicate a high specificity for small restriction fragments of R. meliloti but not for total nif gene sequences. Three linkers, an octa-, deca-, and dodecanucleotide were prepared, all of which contain Hind III restriction sites.