Kazumitsu Onizuka

Pin-point chemical modification of RNA with diverse molecules through the functionality transfer reaction and the copper-catalyzed azide-alkyne cycloaddition reaction.

The internal modification of RNA has been successfully achieved by the functionality transfer reaction (FTR) and following click chemistry with diverse azide compounds. The benefits of the FTR have been demonstrated by its specificity, rapidity, broad applicability, and procedure simplicity.

Site-specific covalent modification of RNA guided by functionality-transfer oligodeoxynucleotides

Efficient methods for the covalent modification of large RNA molecules should find significance utility as innovative biological tools as well as therapeutic methods. In this study, the development of a general method for site-specific RNA modification guided by the functional ODN template has been investigated. The ODN probe containing 6-thioguanosine was modified by the methylenediketone derivative to form the S-functionalized ODN. Site-specific and cytosine-selective RNA modifications were achieved by the functionality-transfer reaction from the sulfur atom of the functionalized probe to the amino group of the cytosine base of the target strand. It was shown that the base and site selectivity were due to the close proximity of the reactants in the DNA-RNA duplexes.

The oligodeoxynucleotide probes for the site-specific modification of RNA

As the knowledge of the biological functions of RNA expands, the demand for research tools to investigate intracellular RNA is increasing. Oligonucleotides can be rationally designed for the target RNA sequence, and therefore, have become a reliable platform for the development of specific molecules for RNA. The chemical modification of RNA has a strong impact on RNA research; the fluorescent labeling of RNA is useful to monitor RNA production, processing, relocation in the cell, interaction with other intracellular components and degradation, etc. Chemical modification may affect the RNA function through a variety of pathways, and therefore, would be potentially useful for biological research, therapeutic approach and artificial manipulation of the RNA function. This tutorial review starts with an introduction of the biological relevance of modified RNA, and focuses on the recent progress of the oligodeoxynucleotide probes for the covalent modifications of RNA. The prospects of this new technology are also discussed.

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.

Activation and Alteration of Base Selectivity by Metal Cations in the Functionality-Transfer Reaction for RNA Modification

Previously, we reported that the 2-methylidene-1,3-diketone unit of 6-thioguanosine transferred selectively to the amino group of cytosine at pH 7.0 and that its selectivity was changed to the guanine base at pH 9.6. In this study, it was found that the functionality-transfer reaction enhanced selectivity for the guanine base in the presence of divalent transition metal cations such as Ni(2+) and Co(2+) at pH 7.4.

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.

A new odorless procedure for the synthesis of 2'-deoxy-6-thioguanosine and its incorporation into oligodeoxynucleotides

6-S-[2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxy-6-thiogua nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl 3-mercapto-propionate. The phosphoramidite precursor derived from 2 was successfully applied to an automated DNA synthesizer to produce 2′-deoxy-6-thioguanosine containing ODN. The results showed that 2-ethylhexyl 3-mercaptopropionate is useful as an odor less reagent and also as an S-protecting group of 2′-deoxy-6-thioguanosine.

Automatic Pseudorotaxane Formation Targeting on Nucleic Acids Using a Pair of Reactive Oligodeoxynucleotides

Here we report a novel method to form a pseudorotaxane architecture using only a pair of reactive oligodeoxyribonucleotides (ODNs), which we designed and synthesized, and then performed the pseudorotaxane formation reaction with both DNA and RNA oligonucleotides. The reaction proceeded smoothly without any extra reagents at 37 °C and pH 7.2, leading to the formation of a stable complex on a denaturing polyacrylamide gel. Interestingly, the pseudorotaxane was formed with the cyclized ODN reversibly by the slipping process. This new pseudorotaxane formation represents a promising method for developing new DNA nanotechnologies and antisense oligonucleotides.

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.

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.