Efficient Thymidine-Selective DNA Interstrand Photo-activated Crosslinking by the 6-Thioguanine Connected via an Ethylene-Linker to the 2'-Deoxyribose Unit.

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 cross-links 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.

An efficient and simple method for site-selective modification of O6-methyl-2'-deoxyguanosine in DNA.

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.

The alkyl-connected 2-amino-6-vinylpurine (AVP) crosslinking agent for improved selectivity to the cytosine base in RNA.

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.

Site-specific modification of RNA by functionality-transfer ODN probes.

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.

Evaluation of the antisense effect of PEGylated oligodeoxynucleotides containing intelligent nucleoside analogues.

We have previously reported that the 2-amino-6-vinylpurine (AVP) in oligonucleotide (ODN) showed the highly selective and effective cross-linking reaction toward cytosine within target complementary sequences. Recently, we revealed that the PEGylated ODN containing AVP analogues exhibited the effective antisense effect in the cultured cell. However, subsequent studies showed that the cross-linking ability of AVP for cytosine was affected by the target RNA sequences. We therefore developed new intelligent nucleoside analogues connected AVP to sugar through the ethylene linker (et-AVP), that exhibited highly effective cross-linking ability to rC in the target RNA. In this paper, we described the synthesis of the PEGylated ODN containing cross-linking nucleoside analogues and properties of it about RNase H activity and antisense effect in cell lysate. As a result, the PEGylated ODN containing et-AVP showed the effective antisense effect in the non-cell system for targeting the luciferase mRNA by non-RNase-H mechanism.

Site-specific modification by functionality-transfer oligonucleotide with the photo-inducible reactivity.

Previously, we reported that S-vinyl thioguanosine analogs exhibited the efficient functionality-transfer reaction with selectivity toward cytidine. This technique was applied to the inhibition and the labelling of DNA or RNA. In this study, we aimed at developing functionality-transfer oligonucleotide with the photo-inducible reactivity. This photo-induced transfer reaction proceeded only to d(m)C but not to dC and dT at the target site of the complementary ODN.