Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector.

A set of single-stranded DNA (ssDNA) expression vectors, which can generate intracellularly any ssDNA or oligodeoxynucleotide (ODN) molecules, have been developed in our laboratory. Studies from our laboratory as well as our collaborators demonstrated that these ssDNA expression vectors are capable of producing: (1) 10-23 DNA enzyme for downregulating c-raf kinase gene expression and (2) triplex-forming oligodeoxynucleotide (TFO) for inducing genomic recombination. We report here the construction of a new version of ssDNA expression vector. A beta-galactosidase (beta-gal) reporter gene was used as a test target so that the alteration of gene expression can be easily measured using beta-gal activity assay. We designed a 10-23 DNA enzyme molecule that specifically cleaves beta-gal mRNA at protein translation starting site (ATG). Using a cell-free RNA cleavage assay, we confirmed that this DNA enzyme molecule could effectively cleave beta-gal RNA. However, a single substitution from T to G in the catalytic domain of this DNA enzyme molecule abolished its RNA cleavage activity. We also constructed an expression vector that can generate DNA enzyme molecules in cells. A549 lung carcinoma cells were cotransfected with both DNA enzyme expression vector and the beta-gal reporter gene. Compared to the cells that were transfected with the mutated DNA enzyme expression vector, significant reduction of beta-gal gene expression (up to 76%) was observed in the cells transfected with DNA enzyme expression vector as indicated by the protein expression level as well as its enzyme activity. These results further suggest that the ssDNA expression vector has potential applications in the study of gene function and target validation.