Naoshi Kojima

A hypothesis-free polymeric sensor array generates fluorescence fingerprints to rapidly identify the type, origin, and quality of complex culture supplements, enabling robust and data-driven quality assessment across diverse biological media.

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.

The advancement of the cell-culture industry toward a circular bioeconomy is driving a growing demand for simple and cost-effective analytical technologies to support the quality control of culture media. Here, we present a hypothesis-free, data-driven sensing strategy that employs an array of synthetic polymer probes to generate fluorescence-response patterns that are able to reflect the comprehensive compositional differences in cell-culture supplements. This approach, based on charged block-copolymers conjugated with aggregation-induced-emission (AIE) fluorophores, can successfully distinguish 16 different animal sera through rapid and simple fluorescence measurements, and can reveal phylogenetic differences among species. Furthermore, the system enables the detection of differences in serum origin, lot, and storage conditions, as well as quality variations in serum-free supplements for stem-cell culture and naturally derived yeast extracts, including subtle changes undetectable by conventional cell-culture assays. As it does not require prior assumptions about which specific components are important, this data-driven strategy can be flexibly adapted to a diverse array of supplement types and quality control needs, offering a robust and reproducible quality assessment with significant potential for standardization that ensures that cell-based products have been reliably manufactured.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.

A biomimetic ‘chemical-nose’ composed of twelve block copolymers with aggregation-induced emission units is presented, which can detect sleep disorder in mice from a small amount of microbiome samples (<10 μg) in a short time (<30 min).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3β-methoxy withaferin-A (3βmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3βmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3βmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 μM and that 3βmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [K_d], 5.9 μM). We propose that 3βmWi-A is a novel modulator of circadian rhythms.

Understanding the status of gut microbiota has been recognized as crucial in health management and disease treatment. To meet the demands of medical and biological applications where rapid evaluation of gut microbiota in limited research environment is essential, we developed new sensing systems able to readout the overall characteristics of complex microbiota. Response patterns generated by a synthetic library of 12 charged block-copolymers with aggregation-induced emission units were analyzed with pattern recognition algorithms, allowing to identify the species/phyla of 16 axenic cultures of intestinal bacterial strains. More importantly, our method clearly classified artificial models of obesity-associated gut microbiota, and further succeeded in detecting sleep disorders in mice through comparative analysis of the normal/abnormal mouse gut microbiota. Our techniques can analyze complex bacterial samples far more quickly, simply and inexpensively than common metagenome-based methods, offering a powerful and complementary tool for gut microbiome analysis for practical use, e.g., in clinical settings.

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 properties of gapmer antisense oligonucleotide (ASO) flanked by deoxyribonucleic guanidine (DNG) were investigated for the potential application in antisense technology. DNG is a unique nucleotide analog which has a positively charged internucleotide guanidinium linkage instead of negatively charged phosphodiester backbone linkage. We prepared a gapmer ASO containing DNG units at both wings of the sequence and compared its properties with 2′,4′-BNA/LNA gapmer ASOs with phosphorothioate (PS) backbone. Although DNG gapmer showed no stabilizing effect on the duplex formation with target RNA, the DNG modification was found to be tolerant to exonuclease digestion. Furthermore, DNG gapmer can induce RNase H-mediated cleavage of target RNA molecule, a requisite property for the antisense strategy. Therefore, the DNG gapmer developed in this study could be an interesting and useful candidate for the development of potent ASOs.