Abstract
We have improved the antisense method for the suppression of specific gene expression by introducing photo-induced binding of antisense oligonucleotides labeled with photo-reactive fluorescein (F-DNA) to a target sequence. Using F-DNA targeted to p53 gene, we have studied 1) in vitro photochemical binding of 18 mer F-DNA to the target 54 mer oligonucleotides including complementary sequence to F-DNA using 15 % polyacrylamide gel electrophoresis and 2) suppression of X-ray or UV-B induced p53 protein in human keratinocytes detected by Western blot. The formation of a complex with higher molecular weight was detected upon visible light irradiation in the in vitro system. Irradiation of F-DNA with no complementary sequence (scramble F-DNA) did not produce such a higher molecular weight band. Cellular experiments showed that irradiation of visible light to cells which incorporated F-DNA induced further reduction of p53 level in addition to the normal antisense effect, while use of scramble F-DNA exhibited little suppression. These results strongly suggest that the present "photo-antisense method" has a potential for biological and medical applications to regulate specific gene function.
