A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.Download high-res image (71KB)Download full-size image

Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We designed three probes consisting of 2-AVP as a reactive moiety and three kinds of binding moiety with high affinity to duplex DNA. Among these probes, Hoechst–AVP probe exhibited high selectivity and efficient reactivity to thymine bases at the site opposite an abasic site in DNA. Our method is potentially useful for inducing site-directed reactions aimed at inhibiting polymerase reactions.

A novel crosslink-forming nucleobase, 2-amino-6-(1-ethylthiovinyl)purine (ATVP), which is triggered by the oxidation of sulfide to sulfoxide, has been developed. The oxidation of ATVP within the duplex proceeded with H2O2 and FeCl2. We have successfully developed the crosslinking reactions activated by oxidation.

Synthetic light-driven molecular motors are molecular machines capable of rotation under photo-irradiation. In this paper, we report the synthesis of peptide-conjugated molecular motors and evaluate their DNA-binding properties.

Oligodeoxynucleotides (ODNs) have been widely used for inhibiting the gene expression in antisense or antigene methods, and the interstrand cross-linking (ICL) forming ODNs have been expected to ensure the inhibition by these methods. Previously, we reported a highly efficient and selective ICL reaction toward cytosine using the 2-amino-6-vinylpurine derivative under acidic conditions. In this Letter, we report the synthesis of ODN containing 6-amino-2-vinylpurine derivatives and evaluation of the cross-linking reactivity.

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position.

Synthetic oligonucleotides (ONs) are valuable tools that interfere with gene expression by specifically binding to target genes in a sequence-specific manner. Reactive ONs containing cross-linking agents are expected to induce efficient inhibition because they bind covalently to target genes. In recent years, researchers have reported several cross-linking reactions that target DNA induced by external stimuli. This short review highlights recently developed novel cross-linking reactions, focusing particularly on nucleoside derivatives developed by our group.

Interstrand cross-linking (ICL) forming oligodeoxynucleotides (ODNs) have been expected to ensure the inhibition of gene expression. In this communication, we report a highly efficient and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-oxopyrimidine derivative.

Non-natural nucleotides with diverse functionalities are highly useful in many areas of basic research and practical applications. We have previously developed an efficient method for post-synthetic modifications of 2-amino-6-vinylpurine (AVP)-containing oligonucleotides, which permits conjugations of a variety of useful functional appendages to the AVP moiety in DNA. Here we report an investigation on the ability of various DNA polymerases to use 5′-triphosphate of 2′-deoxyribosyl-2-amino-6-(2-methylthioethyl)purine (a stable precursor of AVP) as the substrate for templated DNA synthesis.2′-Deoxyribosyl-2-amino-6-(2-methylthioethyl)purine nucleoside 5′-triphosphate (dAVP (SMe)-TP) was examined as a substrate of various DNA polymerases in DNA polymerization reactions to synthesize DNA containing this non-natural functional nucleotide.

Peptide nucleic acids (PNAs) are structural mimics of nucleic acids that form stable hybrids with DNA and RNA. Due to these characteristics, PNAs are widely used as biochemical tools, for example, in antisense/antigene therapy. In this study, we have synthesized PNAs incorporating 2-amino-6-vinylpurine (AVP) for the covalent targeting of single-stranded DNA and RNA, and evaluated their reactivities for these targets. PNA containing AVP at the N-terminal position showed a high reactivity to uracil in RNA and thymine in DNA at the complementary site with AVP. In addition, the crosslinking reactions to pre-miR122 with PNA containing AVP increased the inhibition effect for the Dicer processing of pre-miR122 in vitro.

A novel crosslink-forming nucleobase, 2-amino-6-(1-ethylthiovinyl)purine (ATVP), which is triggered by the oxidation of sulfide to sulfoxide, has been developed. The oxidation of ATVP within the duplex proceeded with H2O2 and FeCl2. We have successfully developed the crosslinking reactions activated by oxidation.

Interstrand cross-linking (ICL) forming oligodeoxynucleotides (ODNs) have been expected to ensure the inhibition of gene expression. In this communication, we report a highly efficient and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-oxopyrimidine derivative.

Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We designed three probes consisting of 2-AVP as a reactive moiety and three kinds of binding moiety with high affinity to duplex DNA. Among these probes, Hoechst–AVP probe exhibited high selectivity and efficient reactivity to thymine bases at the site opposite an abasic site in DNA. Our method is potentially useful for inducing site-directed reactions aimed at inhibiting polymerase reactions.

Synthetic oligonucleotides (ONs) are valuable tools that interfere with gene expression by specifically binding to target genes in a sequence-specific manner. Reactive ONs containing cross-linking agents are expected to induce efficient inhibition because they bind covalently to target genes. In recent years, researchers have reported several cross-linking reactions that target DNA induced by external stimuli. This short review highlights recently developed novel cross-linking reactions, focusing particularly on nucleoside derivatives developed by our group.