Biology subjects

Tajima, K.

Publications and source records attributed to Tajima, K..

10 recordsLinked to original sources

bioRxiv2026

Polymorphic Structures of an Artificial Nucleocapsid Evolved from a Circularly Permuted Bacterial Protein Cage

Molecular self-assembly provides a route to nanoscale structures with defined sizes and shapes. Protein cages assembled from repeated subunits form compartments that can package molecular cargoes. Although amino acid substitutions can alter intersubunit interactions and cage architecture, how protein backbone connectivity relates to the cage architectures accessible during laboratory evolution remains unclear. Here we evolved an artificial nucleocapsid from a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS). Laboratory evolution increased the yield of purified nucleocapsids relative to the progenitor, and the evolved variant, NC119-3, packaged its encoding mRNA. Cryo-electron microscopy revealed a heterogeneous population of cages, within which we resolved 180- and 210-subunit cage families composed of 36 and 42 pentamers, respectively. Within each resolved family, particles with the same subunit stoichiometry differed in the arrangement and orientation of their pentameric building blocks. A 2.90 [A] reconstruction of a tetrahedral 180-subunit cage further revealed that sequence-identical subunits adopt distinct conformations depending on their positions within the assembly. These pentamer-based architectures differ from the domain-swapped T = 4 cage previously reported for nucleocapsids evolved from an AaLS variant with a different circular permutation site. Together with previous work, these findings show that RNA-packaging nucleocapsids evolved from scaffolds with different backbone connectivity can adopt distinct cage architectures.

synthetic biology↗

bioRxiv2026

Efficient cell-free evolution of RNA polymerases by droplet microfluidics

Directed evolution enables the rapid creation of biomolecules with new functions, yet linking genotype and phenotype often requires laborious cell-based workflows. Here we integrate a boosted in vitro transcription-translation system capable of protein expression from single-copy DNA with ultrahigh-throughput fluorescence-activated droplet sorting, developing a simple one-day workflow for efficient protein evolution. Using this system, we evolved SP6 RNA polymerase into a highly robust variant that functions in multiple environments, including cell-free reactions and mammalian cells. We further engineered the evolved enzyme into a proximity-dependent split RNA polymerase that converts molecular interactions into transcriptional activity with minimal background. The resulting biosensors detect diverse molecular targets in vitro, including proteins, peptides, RNAs, and molecular glues. This cell-free platform provides versatile routes for evolving diverse functional biomolecules, accelerating the development of advanced biotechnologies such as diagnostics and therapeutics.

synthetic biology↗

bioRxiv2026

Boosted cell-free gene expression for robust signal readout from a single-copy DNA template in microdroplets

Cell-free gene expression in micro-compartments constitutes a chassis for biotechnology and synthetic biology. Protein synthesis from low concentrations of DNA, a single copy per compartment, is essential for in vitro evolution of biomolecules and synthetic cells. However, insufficient yield of protein synthesized from typically sub-picomolar DNA results in undetectable signals or inadequate activity of desired protein functions. Here we identify and largely mitigate yield-limiting bottlenecks of reconstituted in vitro transcription and translation (IVTT) at low DNA input. Systematic comparison of commercial reconstituted IVTT kits revealed that gene expression starts becoming limited by mRNA scarcity around 20-200 pM DNA input. We further uncovered that the standard ribosome concentration is excessive at low-DNA input and shortens the lifetime of translation. These findings led to a simple optimization recipe that combines supplementation with a highly active T7 RNA polymerase and a reduction in ribosome concentration, which synergistically amplified gene expression by [~]10-fold across diverse fluorescent proteins and enzymes. This low-DNA-optimized formulation in picoliter droplets achieved [~]94 nM protein expression from a single copy of DNA ([~]0.12 pM). The user-friendly boosted IVTT protocol paves the way for straightforward functional screening and in vitro reconstitution of cellular functions in DNA-scarce environments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/707295v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@14fffaborg.highwire.dtl.DTLVardef@11a4bd1org.highwire.dtl.DTLVardef@1679d79org.highwire.dtl.DTLVardef@1937ff6_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Sensitive colorimetric ELISA measuring salivary SBP-specific secretory IgA, its application to pollinosis SLIT

BackgroundSublingual immunotherapy is safe and one of the most effective etiological treatments for pollinosis. Salivary allergen-specific secretory immunoglobulin A (SIgA) is a useful marker to objectively evaluate treatment effectiveness. However, saliva samples usually exhibit high background noise because of nonspecific binding in enzyme-linked immunosorbent assays (ELISAs). ObjectiveWe aimed to establish an amplified ELISA to measure allergen-specific salivary SIgA levels to assess the efficacy of sublingual immunotherapy (SLIT). MethodsBiotinyl tyramide (BT-Tyr) was used to amplify the signal in a colorimetric ELISA. The allergen sugi basic protein (SBP) was biotinylated and used as a reporter to obtain sensitive and specific results with low background. This ELISA was used to measure anti-SBP SIgA levels in saliva samples from SLIT-treated patients with Japanese cedar pollinosis. ResultsThe ELISA was highly sensitive and achieved more than 200-fold signal amplification compared to non-amplified conditions. It was also specific for antigens without background noise. The levels of SBP-specific SIgA in saliva increased in patients 3 months after SLIT initiation. ConclusionWe established a sensitive colorimetric ELISA to measure SBP-specific SIgA in saliva using BT-Tyr-based amplification and biotinylated antigen as a reporter. We used this sensitive ELISA to monitor the levels of SBP-specific SIgA in saliva samples from patients with pollinosis receiving SLIT. Increased salivary SBP-specific SigA levels were observed in these patients.

immunology↗

bioRxiv2025

Cargo-directed assembly of nonviral nucleocapsid with controlled size

Precise packaging of diverse cargo within self-assembling protein cages of defined size and shape is essential for many biotechnological applications, yet cellular expression offers limited control over loading. Here, we developed a system for in vitro cargo-directed reconstitution of a split, laboratory evolved nonviral nucleocapsid (spNC-4). Independently expressed and purified spNC- 4 capsid protein subunits were mixed and assembled with cargo molecules in a cooperative manner. As an authentic cargo, mRNA is packaged into a 30 nm-spheric nucleocapsid in vitro, closely matching to spNC-4 expressed in cells. In this system, a diverse range of cargo molecules, including cognate nucleocapsid mRNA, noncognate RNA, RNA complexed with positively supercharged fluorescent protein, and linear double-stranded DNA are encapsulated within the 30 nm-spheric nucleocapsids in vitro. Moreover, the packaging of 30 nm-spherical or rod-shaped DNA origamis as templates induce morphological alterations of the nucleocapsids, resulting in the formation of enlarged 60 nm-spherical structure or rod-shaped structure, respectively. This split-protein, cargo-dependent system provides versatile and programmable control over both composition and architecture of nonviral protein cages, creating a general platform for enzyme nanoreactors, targeted delivery, and vaccine development.

synthetic biology↗

bioRxiv2025

Modulation of gluconic acid metabolism enhances nanofibrillated bacterial cellulose production from agro-industrial by-products

Efficient and robust bacterial cellulose production is essential for advancing the sustainable bioeconomy. In this study, we investigated the impacts of metabolism of organic acids, mainly gluconic acid (GA), on nanofibrillated bacterial cellulose (NFBC) production by Komagataeibacter intermedius NEDO-01 under various culture conditions in aerated stirred-tank reactors. In cultures of the wild-type strain in a standard medium, rapid GA production decreased the medium pH and depleted glucose, inhibiting cell growth and reducing the NFBC yield. However, proper pH control and continuous feeding reversed these effects, resulting in a 3-fold increase in NFBC yield (from 2.45 to 7.59 g/L). In cultures of a glucose dehydrogenase gene-deficient ({Delta}gcd) strain, lack of a pH drop and glucose depletion facilitated better cell growth, yielding 1.85-times more NFBC than that in wild-type cultures under pH-uncontrolled no-feed conditions (4.53 g/L). Notably, GA supplementation accelerated cell growth but significantly inhibited NFBC synthesis, suggesting that GA uptake redirects the carbon flux toward central metabolism. In the corn steep liquor (Csl)-based medium, cell growth was significantly enhanced, and NFBC yield was equivalent to or higher than that obtained with the Hestrin-Shramm medium. GA accumulation was markedly reduced, suppressing pH fluctuation. Under these optimized conditions, three molasses types were tested with Csl, yielding relatively high NFBC. Structural analysis of NFBC produced using these alternative media revealed slight differences in the fiber width distribution, with crystallinity and fiber width remaining constant. Overall, NFBC of consistent quality can be produced in stirred-tank reactors using Komagataeibacter spp. from various agricultural by-products. ImportanceIn this study, we investigated the interplay between organic acid metabolism and nanofibrillated bacterial cellulose (NFBC) production in stirred-tank reactor (STR) cultures of Komagataeibacter intermedius NEDO-01. While it is well known that gluconic acid production competes with cellulose biosynthesis in Komagataeibacter, the quantitative relationship between these pathways under varying culture conditions has not been fully elucidated. By applying optimized feeding strategies and employing a glucose dehydrogenase knockout mutant, we demonstrated that suppressing gluconic acid accumulation significantly enhances NFBC yield. Furthermore, we explored the use of agro-industrial by-products, including molasses and corn steep liquor, as alternative, low-cost feedstocks. Structural characterization confirmed that NFBCs produced under these conditions maintained consistent quality. These findings contribute to the development of scalable, cost-effective microbial production processes for nanocellulose, which is essential for advancing the sustainable bioeconomy. Key PointsO_LIGA accumulation inhibited growth and cellulose production by Komagataeibacter C_LIO_LIEnhanced central metabolism elevated NFBC yield but reduced its production rate C_LIO_LIConsistent NFBC properties were achieved in STRs using various by-product sources C_LI

bioengineering↗

bioRxiv2023

Cell viability is dominated by quantum effects

Quantum tunneling is a phenomenon in which small quantum particles pass through a reaction energy barrier, as if they were passing through a tunnel opened in the barrier. In this study, we analyzed the involvement of the quantum tunneling effect in enzymatic chemical reactions involving hydrolysis in vitro, by monitoring the kinetic isotope effects due to hydrogen isotopes and their temperature dependence as indicators. The results demonstrated that the quantum tunneling effect is involved in deacetylation, DNA cleavage, and protein cleavage reactions. These related reactions were also examined in terms of their effects on cells, which revealed that the quantum effect is even involved in cell survival, including the almost complete inhibition of DNA homologous recombinational repair. One-Sentence SummaryDue to quantum effects in numerous enzymatic reactions mediated by hydrolysis, heavy water significantly impacts biological outcomes in cells.

biophysics↗

bioRxiv2023

Quantum effects on histone deacetylation and cellular transcription

The question of whether physical phenomena at a quantum level significantly impact aspects of macroscopic life has long remained unanswered. Histone modification by acetylation regulates the transcriptional activity of genes, and thereby broadly impacts cellular metabolism. In chemical reactions, the quantum tunneling effect is a phenomenon in which a small quantum particle of the reactant can pass through the potential energy barrier, even if it does not have sufficient energy to overcome the barrier. Here, we demonstrated that quantum effects are involved in the enzymatic reaction of histone deacetylation, by monitoring kinetic isotope effects due to hydrogen isotopes of water molecules and their temperature dependence as indicators. Due to the kinetic isotope effects associated with the quantum effects, the reaction rate balance between histone acetylation and deacetylation in cells was altered with heavy water, which changed epigenetic transcription regulation in the cells. Thus, microscopic quantum mechanisms exist in histone deacetylation, thereby broadly impacting macroscopic life phenomena through epigenetic regulation. TeaserQuantum effects in enzymatic reaction of histone deacetylation latently influence life phenomena through epigenetic regulation.

biophysics↗

bioRxiv2023

Mechanism Underlying the Immune Responses of a Sublingual Vaccine for SARS-CoV-2 with RBD Antigen and Adjuvant, Poly(I:C) or AddaS03, in Non-human Primates

A sublingual vaccine formulated with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD) antigen and Poly(I:C)) adjuvant was assessed for its safety in non-human primates. This Poly(I:C)-adjuvanted sublingual vaccine was safe compared to the AddaS03-adjuvanted vaccine in blood tests and plasma CRP. The safety of the vaccine was also confirmed through quantitative reverse transcription PCR of six genes and ELISA of four cytokines associated with inflammation and related reactions. The Poly(I:C)- or AddaS03-adjuvanted sublingual vaccine produced RBD-specific IgA antibodies in nasal washings, saliva, and plasma. SARS-CoV-2 neutralizing antibodies were detected in plasma, suggesting that adjuvanted-sublingual vaccines protect against SARS-CoV-2 infection. "Yin and Yang"-like unique transcriptional regulation was observed through DNA microarray analyses of white blood cell RNAs from both vaccines, suppressing and enhancing immune responses and up- or downregulating genes associated with these immune responses. Poly(I:C) adjuvanted sublingual vaccination induced atypical up- or downregulation of genes related to immune suppression or tolerance; Treg differentiation; and T-cell exhaustion. Therefore, Poly(I:C) adjuvant is safe and favorable for sublingual vaccination and can induce a balanced "Yin/Yang" -like effect on immune responses.

immunology↗

bioRxiv2019

Loss of a novel striated muscle-enriched mitochondrial protein Coq10a enhances postnatal cardiac hypertrophic growth

Postnatal mammalian cardiomyocytes undergo a major transition from hyperplasia (increases in cell numbers) to hypertrophy (expansion in cell size). This process is accompanied by rapid mitochondrial biogenesis and metabolic switches to meet the demand of increased cardiac output. Although most mitochondrial components express ubiquitously, recent transcriptomic and proteomic analyses have discovered numerous tissue-specific mitochondrial proteins whose physiological functions are largely unknown. Here we report that a highly evolutionarily conserved mitochondrial protein Coq10a is predominantly expressed in mammalian cardiac and skeletal muscles, and is highly up-regulated around birth in a thyroid hormone-dependent manner. Deletion of Coq10a by CRISPR/Cas9 leads to enhanced cardiac growth after birth. Surprisingly, adult Coq10a mutant mice maintain the hypertrophic heart phenotype with increased levels of coenzyme Q (CoQ) per cardiomyocyte, preserved cardiac contractile function and mitochondrial respiration, which contrasts with reported mice and humans with mutations in other Coq family genes. Further RNA-seq analysis and mitochondrial characterization suggest an increase of mitochondrial biogenesis in the Coq10a mutant heart as a possible consequence of Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC1) activation, consistent with a recent intriguing report that CoQ may function as a natural ligand and partial agonist of Peroxisome Proliferator-Activated Receptor (PPAR) /{gamma}. Taken together, our study reveals a previously unknown function of a novel striated muscle-enriched mitochondrial protein Coq10a in regulating postnatal heart growth.

developmental biology↗