Biology subjects

Furubayashi, T.

Publications and source records attributed to Furubayashi, T..

4 recordsLinked to original sources

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↗

bioRxiv2021

Evolutionary transition from a single RNA replicator to a multiple replicator network

In prebiotic evolution, self-replicating molecules are believed to have evolved into complex living systems by expanding their information and functions open-endedly. Theoretically, such evolutionary complexification could occur through successive appearance of novel replicators that interact with one another to form replication networks. Here we performed long-term evolution experiments using an RNA that replicates by a self-encoded RNA replicase. The RNA diversified into multiple coexisting host and parasite lineages, whose frequencies in the population initially fluctuated and gradually stabilized. The final population, comprising five RNA lineages, forms a replicator network with diverse interactions, including cooperation to help the replication of all other members. These results support the capability of molecular replicators to spontaneously develop complexity through Darwinian evolution, a critical step for the emergence of life.

evolutionary biology↗

bioRxiv2019

Evolutionary arms-races between host and parasitic RNA replicators in an artificial cell-like system

In the prebiotic evolution, molecular self-replicators are considered to develop into diverse, complex living organisms. The appearance of parasitic replicators is believed inevitable in this process. However, the role of parasitic replicators on prebiotic evolution remains elusive. Here, we demonstrated experimental coevolution of RNA self-replicators (host RNAs) and emerging parasitic replicators (parasitic RNAs) for the first time by using an RNA-protein replication system we had developed. During a long-term replication experiment, a clonal population of the host RNA turned into an evolving host-parasite ecosystem through the continuous emergence of new types of host and parasitic RNAs produced by replication errors. The diversified host and parasitic RNAs exhibited evolutionary arms-race dynamics. The parasitic RNA accumulated unique mutations that the host RNA had never acquired, thus adding a new genetic variation to the whole replicator ensemble. These results provide the first experimental evidence that the coevolutionary interplay between host-parasite molecules play a key role in generating diversity and complexity in prebiotic molecular evolution.

evolutionary biology↗