Site-specific modification of the 6-amino group of adenosine in RNA by an interstrand functionality-transfer reaction with an s-functionalized 4-thiothymidine.

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.

Remarkable acceleration of a DNA/RNA inter-strand functionality transfer reaction to modify a cytosine residue: the proximity effect via complexation with a metal cation.

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.

4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA.

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson-Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 5'-side of the opposing uridine. In the absence of a 5'-adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research.

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.