S-oligos

Another interesting observation is that several species of filarial nematodes have been shown to express chitinase (Fuhrman, 1995). Indeed the chitinase of A. viteae infective stage larvae (L3) is the main target of the protective humoral immune response when jirds are vaccinated with irradiated attenuated L3s (Adam et al., 1996 see also Chapter 10). It remains to be established whether there is an interaction between the parasite s oligo-chitin A-glycans and chitinase and whether such an interaction has a role to play in parasite-host interaction. 

Table TDDFT(B3LYP) calculated excitation energies (eV) for oligo(cyclopentadiene)s, oligo(pyrrole)s, oligo(phosphole)s (Ph) ollgo(thlophene)s (Th)n, and the corresponding polymers (DFT HOMO-LUMO gaps in parentheses)...

TOMA 0GM7F CACIA TKGS PCOCk ACACC ACAKT TCCAA TCMG-S oligo 1... 

Data from [8]. Labeled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methyl-phosphonate-phosphodiester (Alt-MP-oligo), and unmodified phosphodiester (D-oligo) oligodeoxynu-cleotides. 

Figure 9.1 Helical conformation of Moore s oligo(meta-phenylene ethynylenejs, 1, and Tew s oligo(ortfio-phenylene ethynylenejs, 2.

As mentioned previously in Section 1, Moore s oligo-phenes, when cyclized (rings formed by different units of mPE), can also form nanotubes (Figure Lee et al. 

X = or 0 -diQstereoisotopomenc oligonucleotides X=OAlkyl(Aryl)-DNA triesters X=S -OligoS... 

Total cellular mRNA from PS-S-Oligo treated cells was obtained by using mRNA Purification Kit (Quick Prep Micro, Amersham Pharmacia Biotech, USA) according to the manufacturer s protocol. The mRNA was stored at -80°C after treatment with DNasel (Takara Shuzo Co., Ltd., Japan) for2hat37°C. 

Figure I. Schematic drawing of the photo-cross-linking between PS-S-Oligo and the target mRNA. A cyclobutane bridge is formed between the pyrone ring of the psoralen and the uracil residue of the target mRNA.

The cross-linking reaction of PS-P-As was evaluated by reversed phase HPLC. The equimolar mixture of PS-P-As and sense-RNA was irradiated with a UVA transilluminator (365nm). Figure 2 shows the time course ofthe production of photoproducts. A new peak (arrow) increased as the irradiation time increased. These results indicate that Ps-S-Oligo has an ability to cross-link efficiently with sense-RNA. 

Prior to cell treatments by Ps-S-Oligo, the growth susceptibility of C4II and SiHa cells against UVA-irradiation (365nm) was evaluated. The irradiation time in this study was decided to be 2min to avoid the cell damage. 

S-Oligos, which do not contain psoralen did not show any inhibitory effect on the growth of C4II and SiHa up to 5pM. Also, Ps-S-Oligo did not show any inhibitory effects without UVA-irradiation. These results indicate that psoralen, which can intercalate between nucleobases and can stabilize the hybrid, did not enhance the inhibitory effect on C4II cell growth without UVA-irradiation. On the other hand, UVA-irradiation did drastically affect the cellular proliferation in the presence of PS-P-As in a concentration-dependent marmer. 

The mechanistic investigation was focused on two aspects. The one is the cellular localization of PS-S-Oligo. The second is the quantitation of mRNA. The fluorescence microscopic image using fluorescein-labeled PS-P-As showed that it was mainly accumulated in the proximity of the nuclei but scarcely inside the nuclei (data not shown). 

RT-PCR studies were done to evaluate the amount of the intact mRNA in the Ps-S-Oligo treated C4II cells. Total mRNAs isolated from cells after Ps-S-Oligo treatment with UVA-irradiation were transcribed by reverse transcriptase in the presence of oligo(dT)i6 primer, followed by PCR. Two sets of PCR primers were designed. The one amplifies the region where PS-... 

Figure 3. Effect of psoralen-conjugated oligo(nucleoside phosphorothioate)s (PS-S-Oligo) on the proliferation of human cervical carcinoma cell, C4II. and SiHa. [A] C4II cell line. PS-P-As without UVA-irradiation (4-), PS-P-scr without UVA-irradiation (A ), PS-P-As with UVA-irradiation ( ), PS-P-scr with UVA-irradiation (A). [B] SiHa cell line. PS-P-As without UVA-irradiation ( ), PS-P-scr without UVA-irradiation (A), PS-P-As with UVA-irradiation ( ), PS-P-scr with UVA-irradiation (A). UVA-irradiation 365nm, I.lmJ/cm sec. Cell growth was evaluated by modified MTT assay.

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