Biology subjects

Terasaka, N.

Publications and source records attributed to Terasaka, N..

7 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

Artificial initiation codons and engineered initiator tRNAs enable N-terminal noncanonical amino acid incorporation in intact cell-free translation systems

Noncanonical amino acid (ncAA) incorporation at the protein N-terminus provides a powerful strategy for installing defined chemical handles while minimizing perturbation of internal protein sequences. However, highly efficient initiation-based ncAA incorporation systems suppress the native methionine pathway by removing methionine or methionyl-tRNA synthetase, limiting their use for proteins containing internal methionine residues. Here, we developed an orthogonal initiation system for selective N-terminal ncAA incorporation into proteins in intact cell-free translation systems. We systematically profiled background initiation from all 64 codons in reconstituted translation systems and identified low-background artificial initiation codons. Engineered initiator tRNAs, termed tRNAIniTx, were then designed to decode selected codons and support ncAA-dependent initiation. The optimized CAC/tRNAIniTx04GUG pair enabled efficient N-terminal incorporation of N-biotinyl-L-phenylalanine without removing methionine or methionyl-tRNA synthetase, reaching over 90% incorporation. The system was further extended to p-azido-L-phenylalanine and to an Escherichia coli extract-based cell-free translation system. Finally, N-terminally biotinylated proteins were directly immobilized on streptavidin biosensors for purification-free biolayer interferometry analysis of computationally designed Brd4BD2 binders. This work establishes a codon-guided orthogonal initiation strategy for N-terminal protein functionalization while preserving the native methionine translation pathway.

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

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

An engineered closed-shell, two-component, 480-subunit nucleocapsid

Self-assembling protein cages are powerful nanoscale containers for biotechnology and medical applications. Two-component systems are especially attractive due to their potential for functional complexity. In this study, we demonstrate that the subunits of the 240mer nucleocapsid NC-4, which was previously evolved to package and protect its encoding mRNA, can be split into two fragments without disrupting cage assembly or structure, generating a two-component, 480-subunit capsid. This modification introduces additional termini on the cages exterior surface, creating new opportunities for functionalization. We exploited these new sites by genetically appending peptide and protein tags to the exterior surface of split NC-4 (spNC-4), enabling site-specific glycosylation via post-translational modification and cell-specific delivery by targeted antibody recruitment. Our findings broaden the utility of the NC-4 nucleocapsid. By extension, splitting related protein compartments that bind diverse cargoes could offer a robust platform for biotechnological applications requiring simultaneous encapsulation and customizable surface modification. SIGNIFICANCE STATEMENTNatural protein shells such as viral capsids and bacterial microcompartments have inspired efforts to design synthetic compartments that protect and deliver functional molecules. Here we show that a nonviral, artificially evolved nucleocapsid can be split into two fragments that reassemble into a closed, two-component, 480-subunit cage that packages its own mRNA. This redesign preserves the original architecture and selective RNA packaging while creating new engineerable sites on both the interior and exterior surfaces, enabling simultaneous control of internal cargo binding and external modification. The resulting two-component architecture highlights the structural plasticity of synthetic nucleocapsids and provides a general strategy for constructing modular, evolvable protein containers for biotechnology and synthetic biology.

biochemistry↗

bioRxiv2020

Evolution of a virus-like architecture and packaging mechanism in arepurposed bacterial protein

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme lacking affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding mRNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid impermeable to nucleases, while emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing non-viral carriers for diverse vaccine and delivery applications.

biochemistry↗