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

The use of antisense oligodeoxynucleotides (AS-ODN) remains a viable method to downregulate selected gene function. However, limitations to the antise nse approach remain, such as (1) difficulties in delivery of the AS-ODN int o 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 stud ies, To circumvent some of these difficulties, we designed a vector set tha t directs the in vivo production of single-stranded DNA (ssDNA) of a desire d target sequence with limited extraneous vector nucleotide sequences. One plasmid was designed to express Moloney murine leukemia virus (MoMuLV) reve rse transcriptase (RT), Another expression plasmid contains the MoMuLV prim er 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 te st this expression system, we constructed a plasmid set, pssXA/pssXB that p roduces ssRNA-cleaving DNA 10-23 enzyme (Santoro, S,W., and Joyce, G.F. [19 97], Proc, Natl, Acad, Sci, USA 37, 13330-13342), The DNA enzyme sequence w as placed between two oligonucleotide arms that are complementary and able to specifically target C-raf kinase mRNA, These plasmids were transfected i nto the A549 lung carcinoma cell line. Reduced C-raf mRNA levels by up to 3 4%-36%, as determined by Northern blot analysis, were observed in the trans fected cells. Our results demonstrate the feasibility of using this novel s sDNA expression system to generate any sequence of interest in vivo for ant isense, RNA-cleavage DNA enzyme, or tripler-forming strategies.