Specific Gene Silencing by Single Stranded Large Circular Antisense Molecules

Abstract

I report that single-stranded antisense as a part of large circular (LC-) genomic DNA of recombinant M13 phage exhibits enhanced stability, sequence specific antisense activity, and no need for target site search. A cDNA fragment (708 bp) of rat TNF-α was inserted into a phagemid vector, and TNF-α antisense molecules (TNFα-LCAS) were produced as single-stranded circular DNA. When introduced into a rat monocyte/macrophage cell line, WRT7/P2, TNFα-LCAS was able to ablate LPS-induced TNF-α mRNA to completion. The antisense effect of TNFα-LCAS was shown to be sequence-specific because expressions of three control genes (β-actin, GAPDH and IL-1β) were not significantly altered by the antisense treatment. Further, TNFα-LCAS was found to be highly efficacious as only 0.1 μg (0.24 nM) of TNFα-LCAS was sufficient to block TNF-α expression in 1×105 WRT7/P2 cells. I have also observed specific antisense activity in reduction of NF-κB gene expression. The results suggest that an antisense sequence as a part of single-stranded circular genomic DNA has a specific antisense activity.