Biology subjects

Bae, W.

Publications and source records attributed to Bae, W..

9 recordsLinked to original sources

bioRxiv2026

Autonomous Homeostatic Synthetic Cells via Self-Gating DNA Nanopores

Homeostasis is a fundamental hallmark of living organisms, arising from the complex interplay between biochemical reactions and regulatory feedback systems. Reconstituting such self-regulating behaviour in minimal synthetic cells enables continuous, persistent operation of biochemical reactions for extended amount of time. In this work, we demonstrate a minimal homeostatic synthetic cell capable of autonomous flux regulation using DNA nanotechnology and bottom-up synthetic biology. Our homeostatic architecture consists of Giant Unilamellar Vesicles (GUVs) equipped with gated DNA nanopores, encapsulated in vitro transcription (IVT) machinery, and an RNA degradation system. We achieve homeostasis under varying external chemical stimuli specifically varying concentrations of rNTPs by implementing a negative feedback loop between rNTP influx and RNA production. In our system, DNA nanopores facilitate the influx of rNTPs from the external environment, driving internal transcription. Crucially, the transcription process generates RNA "blockers" designed to bind and gate the DNA nanopores, thereby attenuating further rNTP influx. Our system is dynamic as encapsulated RNases slowly degrade the RNA blockers, allowing the pores to reopen as blocker concentration goes down. We first characterise the functionality and gating efficiency of the DNA nanopores using both pre-synthesised and in situ produced DNA and RNA blockers. We then demonstrate that rNTP flux through these pores is sufficient to drive IVT within the GUVs. Finally, by integrating these modules, we demonstrate robust homeostasis: the system maintains a steady-state level of RNA production for up to 16 hours. By harnessing the controllability of negative feedback loop, we demonstrate thresholding of the homeostasis level using single-stranded regulator DNA. This work establishes a versatile framework for engineering adaptive and self-sustaining responsive nanomaterials and synthetic cell chassis.

biophysics↗

bioRxiv2025

QuantGUV: Quantifying Encapsulation Efficiency of Small Molecules in GUVs

1Synthetic cells, constructed through the self-assembly of small molecules, are designed to mimic life-like behaviours by encapsulating functional molecules. For such synthetic cells to accurately replicate cellular reactions, it is critical that the concentrations of encapsulated molecules mirror those in living systems, as reaction kinetics and cellular network states are highly sensitive to these concentrations. However, methods for precisely determining encapsulation efficiency in synthetic cells at single cell resolution have been limited. To address this challenge, we developed QuantGUV, a software-driven, image-based analysis method that determines the concentrations of fluorescent molecules encapsulated within giant unilamellar vesicles (GUVs). We used Quant-GUV to measure the encapsulation efficiencies of fluorescent molecules, ranging in size from 0.5 nm to 20 nm. These measurements were conducted on GUVs formed via the water-in-oil emulsion transfer method under various experimental conditions. Using QuantGUV, we have measured the encapsulation efficiencies of three fluorescence molecules, sulforhodamine B, mEGFP and polystyrene bead, in GUVs formed via the water-in-oil emulsion transfer method. The encapsulation efficiencies for polystyrene bead was close to 100% in most of the conditions while sulforhodamine B and mEGFPs encapsulation efficiencies depended on the parameters during the GUV formation such as concentrations of lipids and oil-water ratio during the GUV formation. By providing crucial insights into encapsulation efficiencies, QuantGUV offers a valuable tool to monitor the building quantitative synthetic cell systems with accurately controlled internal environments which is a critical step towards the creation of synthetic cells.

synthetic biology↗

bioRxiv2024

Accurate antibody loop structure prediction enables zero-shot design of target-specific antibodies

Protein loops, characterized by their versatile structures with varying sizes and shapes, can recognize a wide range of targets with high specificity and affinity. The variable loops of the antibody complementarity-determining region (CDR) are particularly crucial for immune responses and therapeutic applications due to their effective target recognition capabilities. Accurate structure prediction of these antibody loops is essential for the efficient in silico design of target-binding antibodies for therapeutic or industrial use. However, predicting antibody loop structures is challenging due to the lack of evolutionary information from related proteins. Thus, a successful ab initio structure prediction method, which operates without structural templates or related sequences, is crucial for the effective design of antibody loop-mediated interactions. This study demonstrates that highly accurate antibody loop structure prediction enables the effective zero-shot design of target-binding antibody loops. The performance of loop design has been shown to depend on the accuracy of ab initio loop structure prediction, as tested with two versions of our design model. The high affinity, diversity, novelty, and specificity of the antibody loops designed with these new methods were validated experimentally on four target proteins.

bioengineering↗

bioRxiv2023

Information propagation through enzyme-free catalytic templating of DNA dimerization with weak product inhibition

Information propagation by sequence-specific, template-catalyzed molecular assembly is the source of the biochemical complexity of living systems. Templating allows the production of thousands of sequence-defined proteins from only 20 distinct building blocks. By contrast, exploitation of this powerful chemical motif is rare in non-biological contexts, particularly in enzyme-free environments, where even the template-catalyzed formation of dimers is a significant challenge. The main obstacle is product inhibition: the tendency of products to bind to their templates more strongly than individual monomers, preventing the effective catalytic templating of longer polymers. Here we present a rationally designed enzyme-free system in which a DNA template catalyzes, with weak competitive product inhibition, the production of sequence-specific DNA dimers. We demonstrate the selective templating of 9 different dimers with high specificity and catalytic turnover. Most importantly, our mechanism demonstrates a rational design principle for engineering information propagation by molecular templating of longer polymers.

bioengineering↗

bioRxiv2023

Overcoming the speed limit of four-way DNA branch migration with bulges in toeholds

Dynamic DNA nanotechnology involves the use of DNA strands to create programmable reaction networks and nanodevices. The key reaction in dynamic DNA nanotechnology is the exchange of DNA strands between different molecular species, which is achieved through three-way and four-way DNA exchange reactions. While both of these reactions have been widely used to build reaction circuits, the four-way exchange reaction has traditionally been slower and less efficient than the three-way reaction. In this paper, we describe a new mechanism to optimise the kinetics of the four-way DNA exchange reaction by adding bulges to the toeholds of the four-way DNA complexes involved in the reaction. These bulges facilitate an alternative branch migration mechanism and destabilise the four-way DNA junction, increasing the branch migration rate and unbinding rate of the four-way exchange reaction, bringing it closer to the kinetics of the three-way reaction. This new mechanism has the potential to expand the field of dynamic DNA nanotechnology by enabling efficient four-way DNA exchange reactions for in vivo applications.

biophysics↗

bioRxiv2021

Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/468831v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@5df108org.highwire.dtl.DTLVardef@17b653borg.highwire.dtl.DTLVardef@33315borg.highwire.dtl.DTLVardef@ed81c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

In situ generation of RNA complexes for synthetic molecular strand displacement circuits in autonomous systems

Synthetic molecular circuits implementing DNA or RNA strand-displacement reactions can be used to build complex systems such as molecular computers and feedback control systems. Despite recent advances, application of nucleic acid-based circuits in vivo remains challenging due to a lack of efficient methods to produce their essential components - multi-stranded complexes known as "gates" - in situ, i.e. in living cells or other autonomous systems. Here, we propose the use of naturally occurring self-cleaving ribozymes to cut a single-stranded RNA transcript into a gate complex of shorter strands, thereby opening new possibilities for the autonomous and continuous production of RNA strands in a stoichiometrically and structurally controlled way.

synthetic biology↗

bioRxiv2020

Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions

Toehold-mediated strand displacement (TMSD) is a nucleic acid-based reaction wherein an invader strand (I) replaces an incumbent strand (N) in a duplex with a target strand (T). TMSD is driven by toeholds, overhanging single-stranded domains in T recognised by I. Although TMSD is responsible for the outstanding potential of dynamic DNA nanotechnology1, 2, TMSD cannot implement templating, the central mechanism by which biological systems generate complex, far-from equilibrium assemblies like RNA or proteins3, 4. Therefore, we introduce handhold-mediated strand displacement (HMSD). Handholds are toehold analogues located in N and capable of implementing templating. We measure the kinetics of 98 different HMSD systems to demonstrate that handholds can accelerate the rate of invader-target (IT) binding by more than 4 orders of magnitude. Furthermore, handholds of moderate length accelerate reactions whilst allowing detachment of the product IT from N. We are thus able to experimentally demonstrate the use of HMSD-based templating to produce highly-specific far-from-equilibrium DNA duplexes.

biophysics↗

bioRxiv2019

Landscape of tumor-infiltrating immune cells in human immunogenic cancers: B cells are inversely associated with CD8T cells, but positively correlated with Treg cells

The composition of tumor-infiltrating immune cells may be a strong predictor of cancer treatment responses and survival outcomes. While B cells have been suggested to suppress T cell cytotoxicity in preclinical studies, it has been less understood whether B cells will exert immune-regulatory roles in human cancers. We explored immune cell landscapes in six human immunogenic cancers, including bladder cancer, head and neck cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, and renal cell carcinoma by calculating gene expression patterns of immune cell-specific metagenes in a total of 2951 cancers. We demonstrated that tumor-infiltrating activated B cells was correlated with regulatory T cell (Treg) infiltration, but had an inverse association with activated CD8 T cell infiltration consistently across all six human cancers. Tumors infiltrated by activated B cells (ActB+ tumors) demonstrated an elevated expression of regulatory cytokines and immune checkpoints, compared to tumors without infiltration by activated B cells (ActB-tumors). Activated B infiltration was not significantly associated with survival outcomes.\n\nPrecisThis human cancer tissue analysis showed that tumor infiltration by activated B cells correlates with decreased infiltration by activated CD8 T cells in immunogenic solid tumors, implicating B cell inhibition may enhance T cell-mediated cytotoxicity.

cancer biology↗