Modified nucleosides in natural RNA molecules are essential for their functions. Non-natural nucleoside analogues have been introduced into RNA to manipulate its structure and function. We have recently developed a new strategy for the in situ modification of RNA based on the functionality transfer reaction between an oligodeoxynucleotide probe and an RNA substrate. 2′-Deoxy-6-thioguanosine (6-thio-dG) was used as the platform to anchor the transfer group. In this study, a pyridinyl vinyl ketone moiety was newly designed as the transfer group with the expectation that a metal cation would form a chelate complex with the pyridinyl-2-keto group. It was demonstrated that the (E)-pyridinyl vinyl keto group was efficiently and specifically transferred to the 4-amino group of the opposing cytosine in RNA in the presence of NiCl2 with more than 200-fold accelerated rate compared with the previous system with the use of the diketo transfer group. Detailed mechanistic studies suggested that NiCl2 forms a bridging complex between the pyridinyl keto moiety and the N7 of the purine residue neighboring the cytosine residue of the RNA substrate to bring the groups in close proximity.
Non-natural RNA modifications have been widely used to study the function and structure of RNA. Expanding the study of RNA further requires versatile and efficient tools for site-specific RNA modification. We recently established a new strategy for the site-specific modification of RNA based on a functionality-transfer reaction between an oligodeoxynucleotide (ODN) probe and an RNA substrate. 2'-Deoxy-6-thioguanosine was used to anchor the transfer group, and the 4-amino group of cytosine or the 2-amino group of guanine was specifically modified. In this study, 2'-deoxy-4-thiothymidine was adopted as a new platform to target the 6-amino group of adenosine. The (E)-pyridinyl vinyl keto transfer group was attached to the 4-thioT in the ODN probe, and it was efficiently and specifically transferred to the 6-amino group of the opposing adenosine in RNA in the presence of CuCl2 . This method expands the available RNA target sites for specific modification.
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...
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