@article {161982, title = {Temperature-Driven Catalytic Switching Enables Sequence-Dependent Amplification of Autocatalytic Ribozymes in Coacervates.}, journal = {Small}, year = {2025}, month = {2025 Nov 18}, pages = {e07108}, abstract = {

Autocatalytic RNA networks confined within coacervate droplets represent minimal systems capable of self-reproduction and compositional variation. While such networks have been studied under static conditions, their behavior under fluctuating environments remains poorly understood. Here, the self-assembly dynamics of the Azoarcus ribozyme is investigated within spermine-polyacrylate coacervates subjected to thermal perturbations. It is shown that temperature acts as a switch for ribozyme catalysis: above a threshold, coacervates support autocatalytic RNA assembly; below it, reaction dynamics are arrested due to diffusion-limited transport. Notably, the switching kinetics are sequence-specific-different ribozyme variants exhibit distinct temperature thresholds for catalytic activity. Reaction rates deviate from Arrhenius behavior inside droplets, highlighting the nontrivial coupling between catalysis and compartmentalization. Finally, it is demonstrated that temperature cycling differentially amplifies ribozyme variants, with selective assembly emerging at intermediate cycling frequencies. These results reveal that fluctuating environments can bias reproduction dynamics in compartmentalized autocatalytic systems, providing a potential route for primitive functional differentiation and\ selection.

}, issn = {1613-6829}, doi = {10.1002/smll.202507108}, author = {Chakraborty, Nayan and Ameta, Sandeep and Thutupalli, Shashi} } @article {139054, title = {Constrained dynamics of DNA oligonucleotides in phase-separated droplets.}, journal = {Biophys J}, year = {2024}, month = {2024 Jan 02}, abstract = {

Understanding the dynamics of biomolecules in complex environments is crucial for elucidating the effect of condensed and heterogeneous environments on their functional properties. A relevant environment-and one that can also be mimicked easily in\ vitro-is that of phase-separated droplets. While phase-separated droplet systems have been shown to compartmentalize a wide range of functional biomolecules, the effects of internal structuration of droplets on the dynamics and mobility of internalized molecules remain poorly understood. Here, we use fluorescence correlation spectroscopy to measure the dynamics of short oligonucleotides encapsulated within two representative kinds of uncharged and charged phase-separated droplets. We find that the internal structuration controls the oligonucleotide dynamics in these droplets, revealed by measuring physical parameters at high spatiotemporal resolution. By varying oligonucleotide length and salt concentrations (and thereby charge screening), we found that the dynamics are significantly affected in the noncharged droplets compared to the charged system. Our work lays the foundation for unraveling and quantifying the physical parameters governing biomolecular transport in the condensed environment.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2023.12.025}, author = {Singh, Anupam and Thutupalli, Shashi and Kumar, Manoj and Ameta, Sandeep} } @article {128092, title = {Multispecies autocatalytic RNA reaction networks in coacervates.}, journal = {Communications Chemistry}, volume = {6}, year = {2023}, month = {2023 May 08}, pages = {91}, abstract = {

Robust localization of self-reproducing autocatalytic chemistries is a key step in the realization of heritable and evolvable chemical systems. While autocatalytic chemical reaction networks already possess attributes such as heritable self-reproduction and evolvability, localizing functional multispecies networks within complex primitive phases, such as coacervates, has remained unexplored. Here, we show the self-reproduction of the Azoarcus ribozyme system within charge-rich coacervates where catalytic ribozymes are produced by the autocatalytic assembly of constituent smaller RNA fragments. We systematically demonstrate the catalytic assembly of active ribozymes within phase-separated coacervates-both in micron-sized droplets as well as in a coalesced macrophase, underscoring the facility of the complex, charge-rich phase to support these reactions in multiple configurations. By constructing multispecies reaction networks, we show that these newly assembled molecules are active, participating both in self- and cross-catalysis within the coacervates. Finally, due to differential molecular transport, these phase-separated compartments endow robustness to the composition of the collectively autocatalytic networks against external perturbations. Altogether, our results establish the formation of multispecies self-reproducing reaction networks in phase-separated compartments which in turn render transient robustness to the network composition.

}, issn = {2399-3669}, doi = {10.1038/s42004-023-00887-5}, author = {Ameta, Sandeep and Kumar, Manoj and Chakraborty, Nayan and Matsubara, Yoshiya J and S, Prashanth and Gandavadi, Dhanush and Thutupalli, Shashi} } @article {89427, title = {Darwinian properties and their trade-offs in autocatalytic RNA reaction networks.}, journal = {Nat Commun}, volume = {12}, year = {2021}, month = {2021 02 08}, pages = {842}, abstract = {

Discovering autocatalytic chemistries that can evolve is a major goal in systems chemistry and a critical step towards understanding the origin of life. Autocatalytic networks have been discovered in various chemistries, but we lack a general understanding of how network topology controls the Darwinian properties of variation, differential reproduction, and heredity, which are mediated by the chemical composition. Using barcoded sequencing and droplet microfluidics, we establish a landscape of thousands of networks of RNAs that catalyze their own formation from fragments, and derive relationships between network topology and chemical composition. We find that strong variations arise from catalytic innovations perturbing weakly connected networks, and that growth increases with global connectivity. These rules imply trade-offs between reproduction and variation, and between compositional persistence and variation along trajectories of network complexification. Overall, connectivity in reaction networks provides a lever to balance variation (to explore chemical states) with reproduction and heredity (persistence being necessary for selection to act), as required for chemical evolution.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-21000-1}, author = {Ameta, Sandeep and Ars{\`e}ne, Simon and Foulon, Sophie and Saudemont, Baptiste and Clifton, Bryce E and Griffiths, Andrew D and Nghe, Philippe} } @article {95781, title = {RNA diversification by a self-reproducing ribozyme revealed by deep sequencing and kinetic modelling.}, journal = {Chem Commun (Camb)}, year = {2021}, month = {2021 Jul 08}, abstract = {

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.

}, issn = {1364-548X}, doi = {10.1039/d1cc02290c}, author = {Jeancolas, Cyrille and Matsubara, Yoshiya J and Vybornyi, Mykhailo and Lambert, Camille N and Blokhuis, Alex and Alline, Thomas and Griffiths, Andrew D and Ameta, Sandeep and Krishna, Sandeep and Nghe, Philippe} } @article {92883, title = {Self-Reproduction and Darwinian Evolution in Autocatalytic Chemical Reaction Systems.}, journal = {Life (Basel)}, volume = {11}, year = {2021}, month = {2021 Apr 01}, abstract = {

Understanding the emergence of life from (primitive) abiotic components has arguably been one of the deepest and yet one of the most elusive scientific questions. Notwithstanding the lack of a clear definition for a living system, it is widely argued that heredity (involving self-reproduction) along with compartmentalization and metabolism are key features that contrast living systems from their non-living counterparts. A minimal living system may be viewed as "a self-sustaining chemical system capable of Darwinian evolution". It has been proposed that autocatalytic sets of chemical reactions (ACSs) could serve as a mechanism to establish chemical compositional identity, heritable self-reproduction, and evolution in a minimal chemical system. Following years of theoretical work, autocatalytic chemical systems have been constructed experimentally using a wide variety of substrates, and most studies, thus far, have focused on the demonstration of chemical self-reproduction under specific conditions. While several recent experimental studies have raised the possibility of carrying out some aspects of experimental evolution using autocatalytic reaction networks, there remain many open challenges. In this review, we start by evaluating theoretical studies of ACSs specifically with a view to establish the conditions required for such chemical systems to exhibit self-reproduction and Darwinian evolution. Then, we follow with an extensive overview of experimental ACS systems and use the theoretically established conditions to critically evaluate these empirical systems for their potential to exhibit Darwinian evolution. We identify various technical and conceptual challenges limiting experimental progress and, finally, conclude with some remarks about open questions.

}, issn = {2075-1729}, doi = {10.3390/life11040308}, author = {Ameta, Sandeep and Matsubara, Yoshiya J and Chakraborty, Nayan and Krishna, Sandeep and Thutupalli, Shashi} }