Presentation Information
[P01-041]Development of an Isothermal DNA Amplification Method for Autonomous Growth of DNA Droplets
○Yusei Kudo1 (1. Institute of Science Tokyo (Japan))
Keywords:
DNA droplet,Artificial cell,Isothermal DNA amplification,non-equilibrium system
The self-replication of living organisms consists of two fundamental processes: growth and division. Biological growth is achieved through the continuous internal synthesis and supply of biomolecules, enabling organisms to maintain themselves as non-equilibrium systems. The ability to sustain this dynamic steady state by constantly turning over molecular components while preserving structural and functional integrity is one of the defining characteristics of life. Reproducing this function in artificial cells is essential for understanding the physicochemical principles of life and a crucial step toward constructing autonomously functioning molecular systems. In recent years, membrane-less artificial cells based on DNA droplets, formed via liquid-liquid phase separation (LLPS), have attracted significant attention due to their programmability. Since these droplets typically form through the self-assembly of DNA nanostructures in thermodynamic equilibrium, their size and quantity are limited by the initial DNA concentration. Realizing autonomous growth of such droplets thus requires a system that continuously amplifies and supplies specific DNA components, driving the system into a non-equilibrium state. In this study, we aimed to establish a foundation for the autonomous growth of DNA droplets by developing a novel isothermal DNA amplification method.
We designed a reaction system employing DNA polymerase for strand extension and RNase H for selective degradation of RNA within RNA-DNA hybrid duplexes, enabling continuous and cyclic generation of the specific single-stranded DNA (ssDNA) required for nanostructure assembly. This system operates under constant temperature conditions compatible with droplet stability.
We successfully demonstrated amplification of target DNA strands from template strands under isothermal conditions. Furthermore, we confirmed that DNA nanostructures assembled from these amplified products retained their phase separation ability, forming DNA droplets comparable to those from chemically synthesized DNA.
Conventional DNA amplification methods such as PCR require thermal cycling with elevated temperatures, under which DNA droplets formed via LLPS would inevitably dissolve. The isothermal method developed here circumvents this limitation, enabling amplification where droplet integrity is maintained. Additionally, ssDNA amplification is governed by the competition between strand extension by DNA polymerase and RNA degradation by RNase H. This competitive balance determines the net ssDNA production rate, suggesting that output can be tuned by adjusting relative enzyme concentrations.
This isothermal amplification method provides a strategy for autonomous self-supply of building blocks in artificial cell-like systems, serving as a key technology for realizing the growth of DNA droplets in a non-equilibrium environment. This approach is expected to pave the way toward artificial cells capable of autonomous growth.
We designed a reaction system employing DNA polymerase for strand extension and RNase H for selective degradation of RNA within RNA-DNA hybrid duplexes, enabling continuous and cyclic generation of the specific single-stranded DNA (ssDNA) required for nanostructure assembly. This system operates under constant temperature conditions compatible with droplet stability.
We successfully demonstrated amplification of target DNA strands from template strands under isothermal conditions. Furthermore, we confirmed that DNA nanostructures assembled from these amplified products retained their phase separation ability, forming DNA droplets comparable to those from chemically synthesized DNA.
Conventional DNA amplification methods such as PCR require thermal cycling with elevated temperatures, under which DNA droplets formed via LLPS would inevitably dissolve. The isothermal method developed here circumvents this limitation, enabling amplification where droplet integrity is maintained. Additionally, ssDNA amplification is governed by the competition between strand extension by DNA polymerase and RNA degradation by RNase H. This competitive balance determines the net ssDNA production rate, suggesting that output can be tuned by adjusting relative enzyme concentrations.
This isothermal amplification method provides a strategy for autonomous self-supply of building blocks in artificial cell-like systems, serving as a key technology for realizing the growth of DNA droplets in a non-equilibrium environment. This approach is expected to pave the way toward artificial cells capable of autonomous growth.
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