We have previously reported that the 2-amino-6-vinylpurine (AVP) nucleoside exhibits a highly efficient and selective crosslinking reaction toward cytosine and displayed an improved antisense inhibition in cultured cells. In this study, we further investigated the alkyl-connected AVP nucleoside analogs for more efficient crosslinking to the cytosine base (rC) of the target RNA. We synthesized three AVP analogs which connect the 2-amino-6-vinylpurine unit to the 2'-deoxyribose through a methylene, an ethylene, or a butylene linker. The ODN incorporating the AVP analog with the methylene or the butylene linker showed a slightly higher crosslinking to the target rC of RNA than the original AVP with no linker. In contrast, the AVP with the ethylene linker formed a selective and efficient crosslink to the rC of the target RNA.
O(6)-Methyl-2'-deoxyguanosine (O(6)-Me-dG) is a mutagenic nucleotide in DNA. O(6)-Me-dG in DNA was rapidly and selectively modified by a functionality transfer reaction using the ODN incorporating 6-S-functionalized thioguanosine. Subsequent labelling of O(6)-Me-dG with the fluorescent FAM or biotin group via click chemistry has permitted the sensitive and selective detection of O(6)-Me-dG in DNA.
Cross-linking is a widely-used technology in the studies of DNA, RNA and their complexes with proteins. Intrinsically active alkylating moieties and photo-activated agents are chemically or enzymatically incorporated into nucleic acids. Thionucleobases resemble the corresponding natural bases, and form crosslinks by UVA irradiation. They form cross-links only with a site in close contact, thereby allowing identification of the contacts within the nucleic acids and/or between the nucleic acids and proteins in complex nucleoprotein assemblies. On the other hand, the thionucleobase forms a cross-link less efficiently for the reaction with the opposite natural base in the DNA duplex. In this study, 6-thioguanine was connected to 2′-deoxyribose through an ethylene linker at the 1′-position (Et-thioG). The linker was expected to bring the 6-thio group close to the nucleobase in the opposite strand. In a duplex in which the 2′-deoxy-6-thioguanosine (6-thio-dG) did not form a crosslink, Et-thioG efficiently formed crosslink with a high selectivity for T by UVA irradiation, but with a much lower efficiency for dA, dG, dC, 5-methyl-dC or dU. Interestingly, the yield of the photo-crosslinked product with dT was effectively improved in the presence of dithiothreitol or sodium hydrosulfide (NaSH) at a low UVA irradiation dose. The efficient and selective cross-link formation at a low UVA dose may be beneficial for the biological application of Et-thioG. Key words 6-thioguanosine; photo cross-link; UVAThe intra-and interstrand cross-linking of nucleic acids is a widely used technology for a variety of purposes; i.e., the induction of DNA damage, regulation and manipulation of genes, fixation of DNA nano-structure, etc. DNA cross-linking agents are chemically or enzymatically incorporated into nucleic acids, which include psoralen, 3-cyanovinylcarbazole, aziridine, quinone methide, aldehyde, abasic site, disulfide, benzophenone, bisamide, alkylating anticancer agents, etc. The 2-amino-6-vinylpurine derivative and 5-methyl-4-vinylpyrimidine-2-one have been developed by our group as unique cross-linking agents.1-4) 6-Thioguanosine, 4-thiothymine and 4-thiouridine are also cross-linking agents, and are characteristic in that they are activated by UVA irradiation to form a cross-link only with the close contact-site either at the nucleic acid or the protein in the complex.5) Because of this unique property, despite little knowledge about the photo-adducts, the thionucleobases have been extensively used to identify the close contact-sites of the nucleic acid. We have applied 6-thio-2′-deoxyguanosine (6-thio-dG) as a platform for the functionality-transfer reaction in the site-specific chemical modification of nucleic acids. [6][7][8] In the meantime, we became interested in the photo-induced interstrand cross-link of 6-thio-dG in the duplex DNA. However, the literature 9) and our own experiments indicated that 6-thio-dG in the oligonucleotide generates little photo-cross-linking with normal bases in the opposite DNA strand. The abs...
Efficient methods for the modification of RNA molecules have been expected as innovative biological tools and therapeutic methods. In this study, the development of a general method for site-specific RNA modification by the functionality-transfer ODN probes has been investigated. Site-specific and cytosine-selective RNA modifications were achieved by the functionality-transfer reaction. It was shown that the base and site-selectivity were due to the close proximity of the reactants in the DNA-RNA duplexes.
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