Asako Yamayoshi

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.

Synthesis of antisense oligonucleotides containing 2'-O-psoralenylmethoxyalkyl adenosine for photodynamic regulation of point mutations in RNA.

2-O-psoralen-conjugated antisense oligonucleotide was able to recognize a point mutation of mRNA. It had outstanding ability to photo-cross-link only to oligoribonucleotides (ORN) having a point mutation. This type of antisense molecule is the only one of its kind so far. To give high photo-cross-linking efficiency and sequence selectivity to antisense molecules, we synthesized novel photo-reactive oligonucleotides (2-Ps-xom) containing psoralen at the 2-O-position adenosine via an ethoxymethylene (2-Ps-eom), propoxymethylene (2-Ps-pom) and butoxymethylene (2-Ps-bom) linker, respectively. We evaluated the photo-cross-linking efficiency and sequence selectivity in photo-cross-linking of 2-Ps-xom to the complementary ORN and to an ORN having a mismatch base. Among them, 2-Ps-eom exhibited superior photo-cross-linking efficiency with high sequence selectivity.

Decoy-DNA Against Special AT-Rich Sequence Binding Protein 1 Inhibits the Growth and Invasive Ability of Human Breast Cancer

Triple-negative (TN) breast cancers, which are characterized by estrogen receptor (-), progesterone receptor (-), and human epidermal growth factor receptor 2 (-), are typically associated with poor prognosis because of their aggressive tumor phenotypes. In recent years, the number of patients with breast cancers has remarkably increased, but there are only few available drugs for treatment of TN breast cancers. The development of novel drugs targeting TN breast cancer is urgently required. In the present study, we focused on the function of special AT-rich sequence binding protein 1 (SATB1) as a target molecule for the treatment of TN breast cancers. By recruiting chromatin remodeling enzymes and transcriptional factors, SATB1 regulates the expression of >1,000 genes related to cell growth and translocation. We synthesized a decoy DNA against SATB1, including the recognition sequence of SATB1. We examined the inhibitory effects of the decoy DNAs on cellular proliferation of a TN metastatic breast cancer cell line (MDA-MB-231). SATB1-decoy DNA inhibited the proliferation of MDA-MB-231 cells. Especially, it was significant that SATB1-decoy DNA drastically reduced the invasive and metastatic capacity of MBA-MB-231 cells. Further, in the case of MCF7 cells (SATB1-negative breast cancer cell line), SATB1-decoy DNA did not exhibit any inhibitory effect. These data suggest that SATB1-decoy DNA may be an effective candidate for use as a molecular-targeting drug for treatment of TN breast cancer.

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.

Specific Regulation of Point-Mutated K-ras-Immortalized Cell Proliferation by a Photodynamic Antisense Strategy

It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2-O-psoralenylmethoxyethyl adenosine (2-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.

Functional regulation of RNA-induced silencing complex by photoreactive oligonucleotides

We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation.

Sequence selective formation of 1,N6-ethenoadenine in DNA by furan-conjugated probe

1,N(6)-Ethenoadenosine derivatives have been applied as fluorescence probes in various fields of biochemistry and molecular biology. We developed a 1,N(6)-ethenoadenosine-forming reaction at a target adenine in DNA duplex and applied it to a mutation diagnosis. Furan-derivatized oligodeoxyribonucleotides were synthesized and fluorescence properties were studied in the presence of complementary strand under oxidative conditions. Strong emissions at 430nm were observed in the presence of the complementary strand with an adenine in front of furan moiety.

Novel photoresponsive cross-linking oligodeoxyribonucleotides having a caged α-chloroaldehyde.

We have developed photoresponsive cross-linking oligodeoxyribonucleotides (ODNs) for sequence-selective interstrand covalent bond formation toward target nucleotides. A phosphoramidite derivative of α-chloroaldehyde whose carbonyl group was converted to a bis(2-nitrobenzyl)acetal group was prepared for the synthesis of photoresponsive α-chloroaldehyde (PCA)-conjugated ODN. The bis(2-nitrobenzyl)acetal group of a PCA-thymidine conjugate was completely removed by UV irradiation at 365 nm (400 mW/cm(2)) for 1 min. Photo-cross-linking studies revealed that PCA-ODN selectively reacted with the target nucleotides having an adenine or a cytosine moiety at the frontal position of the α-chloroaldehyde group.

Cross-Linking Antisense Oligodeoxyribonucleotides with a Photoresponsive α-Chloroaldehyde Moiety for RNA Point Mutations.

Because point mutations in GTPase-coding genes have been reported to be responsible for the transformation of cells, anticancer reagents that react effectively and sequence selectively with target RNAs having a point mutation are highly desired. In this study, we developed novel photo-cross-linking oligodeoxyribonucleotides ((pro)PCA-ODNs) that had a caged α-chloroaldehyde group conjugated to a 2-methylpropanediyl backbone ((pro)PCA) in the middle of the strand. A kinetic study of the deprotection reaction of (pro)PCA-ODN revealed that the bis(2-nitrobenzyl)acetal group was completely deprotected within 1 min. Photo-cross-linking studies of (pro)PCA-ODNs with complementary oligoribonucleotides (ORNs) revealed that (pro)PCA-ODNs reacts efficiently and selectively with the target ORNs that have an adenosine or cytidine residue at a frontal position of the (pro)PCA residue without adverse effects of bases adjacent to the mutation site.

Rate-adjusted cross-linking reaction by photoresponsive α-bromoaldehyde (PBA)-conjugated ODN.

We developed a photoresponsive α-bromoaldehyde-conjugated oligonucleotide (PBA-ODN). The PBA-ODN selectively reacted and formed covalent bonds with target oligonucleotides having adenine or cytosine at the frontal position of the aldehyde derivative. Kinetic studies revealed that PBA-ODN has increased kinetic rates for the formation of cross-linked duplexes compared with the corresponding α-chloroaldehyde-conjugated oligonucleotide (PCA-ODN).