Reiko Iwase

Photodynamic antisense regulation of human cervical carcinoma cell growth using psoralen-conjugated oligo(nucleoside phosphorothioate)

Abstract The antisense strategy has been applied to regulate gene expression in a sequence specific manner, which enables suppression of the proliferation of cancer cells and exploration of the functions of unknown genes. In order to generalize and to enhance the ability of the strategy, functionalization of antisense DNAs was done using a photo-crosslinking reagent, 4,5′,8-trimethylpsoralen, and the possibility of photodynamic antisense regulation of gene expression was examined. Psoralen-conjugated oligo(nucleoside phosphorothioate)s (Ps–S-oligo) were prepared and used to inhibit the proliferation of human cervical carcinoma cells. Upon UVA irradiation of Ps–S-oligo treated cells, Ps–S-oligo complementary to the initiation codon region (Ps–P-As) of HPV18-E6*-mRNA of human cervical carcinoma cells inhibited drastically the cell growth (IC 50 =16 nM). In contrast, Ps–S-oligo with mismatched sequences and scrambled one showed lesser inhibitory effects than Ps–P-As. These results showed that the inhibition by Ps–S-oligo was dependent on (a) sequence, (b) UVA irradiation, (c) concentration and (d) cell line. The amount of intact HPV18-E6*-mRNA was decreased in a sequence dependent manner, indicating that the antiproliferative effect of Ps–P-As was an antisense manner. The psoralen-conjugated antisense DNA has significant potential to regulate gene expression, which may provide useful information to explore the novel gene regulating reagents.

Selective Photo-Cross-Linking of 2′-O-Psoralen-Conjugated Oligonucleotide with Rnas Having Point Mutations

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop a new type of anticancer drugs. In this report, we present a new type of antisense molecule that photo-cross-links to an oligoribonucleotide having a point mutation site in a sequence specific manner. 2′-O-psoralen-conjugated adenosine was synthesized in four steps from adenosine and introduced in the middle of an oligodeoxyribonucleotide (2′-Ps-oligo). Compared with 5′-O-psoralen-conjugated oligodeoxyribonucleotide (5′-Ps-oligo), which has a psoralen at the 5′-end, 2′-Ps-oligo more selectively photo-cross-linked to a pyrimidine base of the site of alteration from purine to pyrimidine in the oligoribonucleotide.

Synthesis of capped oligoribonucleotides by use of protected 7-methylguanosine 5′-diphosphate derivatives

This paper describes the capping reaction of 5′-phosphorylated oligoribonucleotides protected only by a tetrahydropyranyl group at each 2′-position. By use of the conventional capping agent PhSppm7GTMTrmM, m7G5pppGUUA and m7G5′pppGUAUUA were synthesized from the corresponding oligoribonucleotide 5′-monophosphate derivatives. Since the latter was obtained in low yield because of poor solubility of the partially protected pUUA in DMF, the oligomer component was converted into a phosphorimidazolidate species which was found to be soluble in DMF and allowed to react with a newly designed capping agent, ppm7GTMTrmM. This triphosphate bond formation proceeded smoothly to give m7G5′pppGUAUUA in good yield.

Psoralen-conjugated oligonucleotide with hairpin structure as a novel photo-sensitive antisense molecule

Hairpin type psoralen-cojugated oligonucleotides cross-linked with RNA when they hybridized with a perfectly complementary RNA.