Yasuo Komatsu

Chemical ligation of RNA fragments is an effective method for synthesizing long RNAs. It is particularly useful for incorporating site‐specific modifications into long RNAs; however, its low reaction efficiency remains a major challenge. Herein, 5′‐amino‐2′‐substituted uridine or cytidine nucleotides are synthesized, which are attached to the 5′ end of a synthetic RNA and conjugated with a phosphate group at the 3′ end of an alternative RNA fragment, to perform a head‐to‐tail‐type RNA ligation. It is examined how the 2′ position of nucleotides with 5′‐amino or 3′‐phosphate groups in the ligated site affects the ligation reaction. The 2′‐fluoro‐5′‐amino‐nucleotide shows enhanced reactivity compared with the 2′‐ribo‐ or 2′‐deoxy‐5′‐amino‐nucleotide. In contrast, the 2′‐ O ‐methyl modification demonstrates optimal efficacy with the 3'‐phosphate nucleotide. Interestingly, the 2′‐fluoro‐5′‐amino nucleotide maintains remarkable reactivity, even under acidic conditions. A pre‐miRNA comprising 84 nucleotides is synthesized through ligation, and the intracellular functionality of the ligated RNA is confirmed. This study elucidates the effect of 2′ substituents on chemical ligation reactions, providing insights into the chemical synthesis of long RNAs.

DNA polymerases frequently misincorporate ribonucleoside 5′-triphosphates into nascent DNA strands. This study examined the effects of an incorporated ribonucleoside on untargeted mutations in human cells. Riboguanosine (rG) was introduced into the downstream region of the supF gene to preferentially detect the untargeted mutations. The plasmid containing rG was transfected into U2OS cells and the replicated DNA was recovered after 48 h. The mutation analysis using the indicator Escherichia coli RF01 strain showed the frequent induction of untargeted base substitutions at the G bases of 5′-GpA-3′ dinucleotides, similar to action-at-a-distance mutations induced by an oxidatively damaged base, 8-oxo-7,8-dihydroguanine, and an apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) cytosine deaminase. APOBEC3B was then knocked down by RNA interference and the plasmid bearing rG was introduced into the knockdown cells. The untargeted mutations at 5′-GpA-3′ sites were reduced by ~80%. These results suggested that ribonucleosides embedded in DNA induce base substitution mutations at G bases in the same strand by an APOBEC3B-dependent mechanism, implying that ribonucleosides contribute to APOBEC3-dependent cancer initiation events.

An interstrand cross-linked duplex (CD) modification promoted antisense oligonucleotide to be localized in the cytoplasm, resulting in effective knockdown microRNA in cytoplasm. In contrast, single-stranded antisense was confined in the nucleus.

MicroRNAs (miRNAs) regulate cancer cell proliferation by binding directly to the untranslated regions of messenger RNA (mRNA). MicroRNA-148a (miR-148a) is expressed at low levels in breast cancer (BC). However, little attention has been paid to the sequestration of miR-148a. Here, we performed a knockdown of miR-148a using anti-miRNA oligonucleotides (AMOs) and investigated the effect on BC cell proliferation. BC cell proliferation was significantly suppressed by AMO flanked by interstrand cross-linked duplexes (CL-AMO), whereas single-stranded and commercially available AMOs had no effect. The suppression was caused by sequestering specifically miR-148a. Indeed, miR-148b, another member of the miR-148 family, was not affected. Importantly, the downregulation of miR-148a induced a greater and longer-lasting inhibition of BC cell proliferation than the targeting of oncogenic microRNA-21 (miR-21) did. We identified thioredoxin-interacting protein (TXNIP), a tumor suppressor gene, as a target of miR-148a and showed that CL-AMO provoked an increase in TXNIP mRNA expression. This study provide evidence that lowly expressed miRNAs such as miR-148a have an oncogenic function and might be a promising target for cancer treatment.

Human tRNA^His guanylyltransferase (HsThg1) catalyzes the 3′–5′ addition of guanosine triphosphate (GTP) to the 5′-end (−1 position) of tRNA^His, producing mature tRNA^His. In human cells, cytoplasmic and mitochondrial tRNA^His have adenine (A) or cytidine (C), respectively, opposite to G₋₁. Little attention has been paid to the structural requirements of incoming GTP in 3′–5′ nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson–Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5′-end of tRNA^His with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNA^His. Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5′-terminal modification of RNAs.

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody–drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single-domain VHH (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product—single-stranded DNA conjugated at the carboxyl terminus of VHH—retained its affinity for EGFR. To investigate whether this VHH–single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100–500 bp DNA. We confirmed the amplification of the VHH–double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.

The tetrahydrofuran-type abasic site analog (THF) induces large deletion mutations in human cells. To compare the large deletions induced by THF on leading and lagging strand templates, plasmid DNAs bearing the analog at a specific position outside the supF gene were introduced into human U2OS cells. The replicated DNAs recovered from the transfected cells were electroporated into an Escherichia coli indicator strain. THF on the lagging strand template produced more supF mutants than THF on the leading strand template. This unequal mutagenicity was due to the higher frequencies of not only large deletions but also untargeted base substitutions induced in the gene. These results suggested that both types of mutations occur more frequently when abasic sites are formed on the lagging strand template.

Breast cancer is the most frequent cancer affecting women worldwide. Traditional chemotherapy, hormone therapy, and targeted therapy are used for breast cancer treatment. However, breast cancer is a heterogeneous disease, and patients often develop drug resistance. Therefore, various new therapeutic strategies have been investigated, including microRNA regulation. Anti-microRNA oligonucleotides (AMOs) are one of the most potent agents in oligonucleotide therapy. The inhibition activity of an AMO can be increased by flanking its single-stranded antisense sequence (the widely used structure for AMOs) with interstrand cross-linked duplexes (CLDs). An extrastable CLD improves nuclease resistance and stabilizes hybridization with a target. This study investigated the effects of anti-microRNA-21 (miR-21) AMO modified with CLDs on breast cancer cells without using reporter assay. The CLD-modified AMO suppressed breast cancer cell proliferation for a long duration compared to other types of AMOs. In addition, it expectedly up-regulated the miR-21-controlled expression of tumor suppressor genes. Therefore, an AMO flanked by CLDs can be a promising strategy for breast cancer treatment.

We studied a dissolved oxygen sensor in aqueous media based on a mesoporous Pt microelectrode with the aim of obtaining a large sensor output. Mesoporous Pt structures were fabricated via Pt thin film deposition on a skeleton of nanoporous gold. The dissolved oxygen was measured via adoptive stripping voltammetry, i.e., the dissolved oxygen was adsorped on the surface of the mesostructured platinum microelectrode over a certain period. Accumulated oxygen on the surface of the mesostructures was reduced through voltammetry with a large current. This led to a linear increase in the reduction current as a function of the active surface area of the Pt mesostructures, indicating that the entire surface area of the mesostructures is utilized to adsorb and reduce oxygen in the given experimental condition. The proposed method could obtain a lager sensor output compared with sensors under diffusion-controlled reactions and better fits for oxygen sensors in micro total analysis systems.

Abasic sites are formed spontaneously and by nucleobase chemical modifications and base excision repair. A chemically stable abasic site analog was site-specifically introduced into replicable plasmid DNAs, which were transfected into human U2OS cells. The amplified DNAs were recovered from the cells and used for the transformation of a bacterial indicator strain. Large deletion mutations were induced by the analog, in addition to point mutations at the modified site. No apparent sequence homology at the deletion junctions was found. These results suggested that the large deletions induced by the abasic site analog are formed by homology-independent events.