In vivo expression of single-stranded DNA in mammalian cells with DNA enzyme sequences targeted to C-raf.

Abstract

The use of antisense oligodeoxynucleotides (AS-ODN) remains a viable method to downregulate selected gene function. However, limitations to the antisense approach remain, such as (1) difficulties in delivery of the AS-ODN into target tissues, (2) instability of AS-ODN in vivo, (3) uncertanties about the precise mode of action, and (4) toxic effects in animal and human studies. To circumvent some of these difficulties, we designed a vector set that directs the in vivo production of single-stranded DNA (ssDNA) of a desired target sequence with limited extraneous vector nucleotide sequences. One plasmid was designed to express Moloney murine leukemia virus (MoMuLV) reverse transcriptase (RT). Another expression plasmid contains the MoMuLV primer binding site at the 3'-end of its RNA transcript so that an ssDNA would be synthesized by RT when both plasmids are cotransfected into cells. To test this expression system, we constructed a plasmid set, pssXA/pssXB that produces ssRNA-cleaving DNA 10-23 enzyme (Santoro, S.W., and Joyce, G.F. [1997]. Proc. Natl. Acad. Sci. USA 37, 13330-13342). The DNA enzyme sequence was placed between two oligonucleotide arms that are complementary and able to specifically target C-raf kinase mRNA. These plasmids were transfected into the A549 lung carcinoma cell line. Reduced C-raf mRNA levels by up to 34%-36%, as determined by Northern blot analysis, were observed in the transfected cells. Our results demonstrate the feasibility of using this novel ssDNA expression system to generate any sequence of interest in vivo for antisense, RNA-cleavage DNA enzyme, or triplex-forming strategies.