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Self-replication and exponential growth are essential to all living things, the driving force for Darwinian evolution, and potentially useful in nanotechnology for large-scale production of nanoscopic materials. An artificial (nonliving) self-replication system has been shown to exhibit exponential growth and selection using DNA monomer origami tiles templated on a dimer seed. That system purposefully avoided the use of enzymes to get a hint of how self-replication might have evolved in a prebiotic world by using CNVK and UV light to crosslink complementary DNA single strands. For further investigations into competition and extinction and for potential applications involving biocompatibility, we wanted to investigate enzymatic ligation to replace the chemical photo crosslinking step. Here, we present a system which uses thermotolerant T4 DNA ligase and no UV. This system has several additional advantages including a much faster cycling time, yielding 2,000,000 amplifications in 12 h. We also introduce competition to study the possibility of Darwinian-like evolution. Two pairs of DNA origami tiles compete for the same connection strands and show different growth rates under different connection strand concentrations. This system has the potential to combine with other enzymes, such as RNA polymerase to support feedback, allowing us to fine-tune replication dynamics and achieve sophisticated, life-like behaviors. The highly efficient self-replication and exponential growth of DNA origami dimers demonstrated in this work not only enhances our understanding of Darwinian evolution in nature but also opens the door to applications ranging from synthetic biology to smart materials.
Self-replication of species under selective pressures is crucial in biological evolution and diversity. Despite some successes with artificial selection and replication DNA systems, constructing a self-replication system under temperature selective pressure remains largely out of reach. In this work, we presented a temperature-responsive DNA origami system that selectively replicates a seed pattern in each replication cycle. Two dimer species with different conformations were constructed by incorporating a temperature-responsive module in the origami system (dimer AB at 25 °C and dimer AC at 45 °C). The dimer template serves as a seed to crystallize a ladder-like structure on cooling. The ladder structures were then cross-linked using T4 ligase and heated to yield the offspring dimers with identical conformation. The replication of temperature-selected origami dimers was demonstrated by agarose gel electrophoresis, AFM images, and fluorescence measurements. The proposed temperature-selective replication system contributes to fundamental studies of selection and evolution, as well as the design, fabrication, and directed evolution of nanomaterials.
Many new structures, machines, active materials, and devices have been produced in the rapidly growing field of DNA nanotechnology. However, the thermodynamics of complex DNA assemblies remains not well understood. Here, we treat the assembly, melting, and activation of more complex structures where interactions are generally cooperative. We abandon a rigorous theory involving a complex landscape in favor of a two-state (open-close) scenario with effective concentration and entropy effects. We test our model with self-replication of DNA origami motifs of increasing sizes and with FRET on a single DNA pair held in proximity, and it predicts melting temperatures to ~2°C where there is a 50°C shift arising from cooperativity. The model is especially useful for designing interactions between large objects programmed to open and close and is readily adaptable to assembly and activation in systems other than DNA.
The emergence, organization, and persistence of cellular life are the result of the functional integration of metabolic and genetic networks. Here, we engineer phospholipid vesicles that can operate three essential functions, namely transcription-translation of a partial genome, self-replication of this DNA program, and membrane synthesis. The synthetic genome encodes six proteins, and its compartmentalized expression produces active liposomes with distinct phenotypes demonstrating successful module integration. Our results reveal that genetic factors exert a stronger control over DNA replication and membrane synthesis than metabolic crosstalk or module co-activity. By showing how genetically encoded functions derived from different species can be integrated in liposome compartments, our work opens avenues for the construction of autonomous and evolving synthetic cells.
Sensitive and specific detection of luteinizing hormone (LH) is critical for the early diagnosis of diseases and cancers, offering valuable insights for clinical strategies aimed at regulating physiological functions to maintain health. To this end, we developed a novel sensing platform for the ultrasensitive detection of LH, harnessing a mutually orthogonal cascade amplification composed of DNAzyme and self-replicating catalytical hairpin assembly (SR-CHA). Upon binding of LH to its aptamer, an LH/aptamer complex is formed, leading to the release of an initiator strand ("strand A") that triggers the assembly of Y-shaped DNA structures. Each Y-shaped structure comprises a modular DNAzyme-based amplification unit, which generates a one-to-many fluorescence signal, and an initiator mimic sequence capable of self-replicating to produce numerous new initiators for accelerating the formation of additional Y-shaped structures, thereby amplifying the detection signal. This synergistic design resulted in outstanding analytical performance, achieving a detection limit as low as 0.00038 mIU/mL in buffer and 0.0006 mIU/mL in real samples, with a broad dynamic range from 0.001 to 100 mIU/mL. In addition, the proposed method also demonstrated excellent selectivity, and a strong correlation with the standard ELISA method, which reveals its promising potential as a universal simple and sensitive methodology for the construction of various aptamer-based bioassays.
The aim of this study is to explore how porcine epidemic diarrhea virus (PEDV) infection induces reprogramming of glucose metabolism in host cells and its impact on viral replication. We designed a control group and an infection group [infection of porcine intestinal epithelial cells (IPEC-J2) with PEDV]. First, we determined the infection time and dose of the virus by observing the PEDV titer and the expression of the N protein. Then, through proteomic comparative analysis, we studied the enriched differentially expressed proteins, key proteins, and key metabolic pathways in PEDV-infected cells. RT-qPCR and Western blotting were employed to verify the protein or gene expression of key enzymes in the glycolysis and tricarboxylic acid (TCA) cycle pathways in PEDV-infected cells. Finally, we clarified the impact of PEDV-induced glycolytic changes on viral replication by measuring the content of glucose, ATP, and lactic acid, as well as the expression of glucose transporters (SGLT-1 and GLUT-2) and PEDV N protein in cells. The results indicated that the optimal infection time of PEDV in IPEC-J2 was 48 h and the optimal multiplicity of infection was 1. Proteomics results showed that 342 differentially expressed proteins were screened out and mainly enriched in pathways such as glycolysis, digestion and absorption of carbohydrates, and lipid metabolism. PEDV infection upregulated the protein levels of key glycolysis enzymes HKII (P<0.05), LDHA, and PKM (P<0.01) in IPEC-J2 and the gene transcription level of PFK (P<0.01), while downregulating the gene transcription levels of key enzymes CS, OGDH, and IDH in the TCA cycle pathway (P<0.01). In addition, PEDV infection increased intracellular lactate content (P<0.01), decreased the ATP content (P<0.01), upregulated the expression levels of SGLT-1 and GLUT-2 (P<0.05, P<0.01). Pre-treatment of IPEC-J2 with the glycolysis inhibitor 2-deoxy- d-glucose (2-DG) reduced the intracellular expression of PEDV N protein (P<0.05). In conclusion, PEDV infection can induce reprogramming of glucose metabolism in host cells and enhance the replication of the virus. This is manifested as the activation of the glycolysis pathway and the obstruction of the TCA cycle and oxidative phosphorylation. PEDV promotes glucose uptake and up-regulate the expression of key enzymes in the glycolysis pathway to induce reprogramming of glucose metabolism, thus promote its efficient replication in host cells. This study confirms that PEDV infection can induce reprogramming of host cell glucose metabolism and promote viral replication by activating aerobic glycolysis, providing new insights and approaches for the targeted treatment and prevention of PEDV infection. 为探究猪流行性腹泻病毒感染如何诱发宿主细胞葡萄糖代谢的重编程以及对其复制的作用，本研究通过猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)感染猪小肠上皮细胞(intestinal porcine epithelial cell-J2, IPEC-J2)建立宿主细胞模型，并分别设置对照组和感染组。首先通过观察PEDV感染滴度和N蛋白的表达来明确病毒的感染时间和剂量;然后通过蛋白组学比较分析PEDV感染细胞中富集的差异蛋白、关键蛋白以及关键代谢通路等;通过RT-qPCR和Western blotting验证PEDV感染细胞中糖酵解和三羧酸循环(tricarboxylic acid cycle, TCA cycle)途径中关键酶的蛋白或基因表达;最后通过检测细胞中葡萄糖、ATP、乳酸的含量以及葡萄糖转运蛋白SGLT-1、GLUT-2和PEDV N蛋白的表达来明确PEDV诱导的糖酵解变化对其复制的影响。结果表明，PEDV感染IPEC-J2的最佳时间为48 h，感染复数(multiplicity of infection, MOI)为1。蛋白组学结果显示有342个差异蛋白被筛选出来并且主要富集在糖酵解、碳水化合物的消化和吸收以及脂质代谢等途径。其中，PEDV感染显著或极显著上调IPEC-J2中糖酵解关键酶HKII (P<0.05)、LDHA、PKM (P<0.01)的蛋白表达水平，极显著上调PFK的编码基因转录水平(P<0.01);而三羧酸循环途径关键酶CS、OGDH、IDH编码基因的转录水平均极显著下调(P<0.01)。PEDV感染还导致细胞内的乳酸含量显著升高(P<0.01)，ATP含量极显著降低(P<0.01);葡萄糖转运蛋白SGLT-1和GLUT-2的表达水平分别显著、极显著上调(P<0.05、P<0.01)。糖酵解抑制剂2-脱氧- d-葡萄糖(2-deoxy- d-glucose, 2-DG)预处理IPEC-J2能够显著降低胞内PEDV N蛋白的表达(P<0.05)。本研究表明，PEDV感染可以诱发宿主细胞发生葡萄糖重编程并增强病毒的复制能力，表现为糖酵解途径激活，TCA循环及氧化磷酸化作用受阻。其机制是通过促进葡萄糖摄取、上调糖酵解途径关键酶的表达，并通过诱导这种糖代谢的重编程变化，促进其在宿主细胞中的高效复制。本研究证实PEDV感染可诱导宿主细胞葡萄糖代谢重编程，通过激活有氧糖酵解促进自身复制，为靶向治疗和预防PEDV感染提供了新的思路和途径。.
A defining feature of living cells is their ability to self-replicate; but creating artificial cells with this capability remains challenging, due to the complexity of biological division machinery. Rather than seeking to reconstitute this machinery, direct control of DNA replication and compartment division using digital microfluidics (DMF). This approach allows us to precisely orchestrate these two fundamental processes, providing insight into how they must be coupled for successful self-replication. The system achieves controlled cycles of replication and division, with daughter compartments inheriting parental DNA and maintaining genetic continuity across multiple generations - a key feature of living systems that has been difficult to achieve in artificial cells. By implementing these processes through direct physical manipulation rather than biochemical complexity, a simple testbed is provided that will help to disentangle the essential requirements for self-replicating systems.
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Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) has been widely used as a biocontrol agent against Helicoverpa armigera in China. To understand the relationship between the metabolic response in H. armigera and MbMNPV infection, we analyzed the overall metabolic changes occurring in H. armigera fat bodies during MbMNPV infection. The metabolomic data identified 135 differentially expressed metabolites (DEMs) in the MbMNPV-infected group. Based on the metabolomic data, the changes in phosphatidylcholine levels during the entire infection period and its effect on virus replication were determined. The results showed that virus infection led to a significant upregulation of phosphatidylcholine in H. armigera. Phosphatidylcholine supplementation shortened the median lethal time (LT50) and reduced the weight of MbMNPV-infected H. armigera. To further explore the relationship between virus infection and phosphatidylcholine synthesis, we measured the expression of genes related to phosphatidylcholine metabolism and knocked down the significantly upregulated gene LPCAT5 using RNA interference. LPCAT5 knockdown reduced MbMNPV replication by impairing the biosynthesis of phosphatidylcholine. In addition, MbMNPV infection also affected the central carbon metabolic pathway, which is indirectly related to phosphatidylcholine synthesis. This study shows that MbMNPV infection can promote its own infection process by upregulating the synthesis of phosphatidylcholine, providing new insight into the interactions between MbMNPV and H. armigera.
Autonomous self-reproduction is a major goal of bottom-up synthetic biology aimed at building artificial cells. This requires that the genome be replicated by its self-encoded replication machinery. While the reconstituted Escherichia coli chromosomal replication system, termed the Replication-Cycle Reaction (RCR) system, offers a promising platform for genome-scale replication, its generation from genetic information has not yet been achieved. Here we show that a 53 kb circular DNA, termed RCR module-genome, encoding all 26 RCR proteins, can self-replicate in a one-pot reaction when expressed using the protein synthesis using recombinant elements (PURE) system. We first built a prototype of the RCR module-genome and then optimized reaction conditions and solved expression bottlenecks to achieve robust self-replication. This artificial module-genome supports more than 28 doublings of recursive self-replication. This system, termed PRIMES (PURE-driven RCR for In-vitro Module-gEnome Self-replication), represents a milestone toward constructing self-reproducing artificial cells.
Catalysis of bond-forming reactions is key to the development of life-like chemical systems as it allows to build up new material, increasing molecular complexity and diversity. Integrating catalysis with other characteristic properties of life, like self-replication, represents an important advance in the transition from chemistry to life. We have previously shown that catalysis can emerge in synthetic self-replicators that form through supramolecular assembly. However, the organocatalyzed reactions were solely bond-breaking so far. We now report the successful expansion of the catalytic promiscuity of these systems to bond-forming reactions. We show that a self-replicator efficiently catalyzes acyl hydrazone formation between different hydrazides and aldehydes. This marks an important step towards the further development of evolvable systems that combine metabolic activity with self-replication.
DNA is an ideal medium for information storage, offering ultra-high density and long-term stability, making it a promising solution to the global data explosion. However, conventional in vitro DNA storage systems remain constrained by their static nature, limiting dynamic data writing, updating, or self-replication. Inspired by genetic memory and stimulus-responsive behaviors of living cells, in vivo DNA storage leverages cells as both storage and processing units, translating cellular information behaviors into engineered storage architectures for adaptive and autonomous data management. This review systematically outlines the development of in vivo DNA storage, focusing on two core strategies: synthetic information-based storage, which achieves long-term data preservation through DNA encoding and cellular self-replication in an inheritance-mimicking manner, and memory-based digital recording, which enables real-time DNA writing and updating in response to artificial stimuli via a memory-mimicking route. We further discuss how functional integration-such as dynamic data updating, encryption, and logical operations-can expand the versatility of in vivo biological data systems toward cell-inspired information storage and computation. Finally, we address current challenges in storage density, editing efficiency, and cellular stability, and propose future directions for next-generation in vivo DNA storage platforms.
The emergence of a chemical system capable of self-replication and evolution is a critical event in the origin of life. RNA polymerase ribozymes can replicate RNA, but their large size and structural complexity impede self-replication and preclude their spontaneous emergence. Here, we describe QT45, a 45-nucleotide polymerase ribozyme, discovered from random sequence pools, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate (triplet) substrates in mildly alkaline eutectic ice. QT45 can synthesize both its complementary strand using a random triplet pool at 94.1% per-nucleotide fidelity and a copy of itself using defined substrates, both with yields of ~0.2% in 72 days. The discovery of polymerase activity in a small RNA motif suggests that polymerase ribozymes are more abundant in RNA sequence space than previously thought.
Transposable elements (TEs) are mobile DNA sequences capable of self-replication (especially retrotransposons) within the genome, which may lead to various forms of DNA damage. The introduction of this review encompasses the diverse classes and subclasses of TEs, particularly emphasizing the most active TEs present in the human genome. An analysis of the retrotransposition process of TEs is presented, illustrating how this mechanism can result in DNA damage and gene rearrangements. Furthermore, the review meticulously examines the implications of TE insertions on gene expression and genomic organization, which may contribute to the development of various diseases, including cancer. The relationship between TE activation and the aging process is also explored, with an emphasis on that epigenetic modifications associated with aging can lead to the derepression of TEs, thereby promoting genomic instability and inflammation. These factors may play a significant role in the pathogenesis of age-related diseases, such as cancer, cardiovascular disorders, and neurodegenerative conditions. Finally, the review considers potential therapeutic approaches aimed at targeting TE activity to alleviate the impacts of aging and associated diseases.
BACKGROUND: Photosynthetic microorganisms, such as cyanobacteria, are promising candidates for sustainable production of chemicals. Photosynthesis is a unique process where light energy is used to convert CO2 into carbon metabolites that sustain the cell`s metabolism. One of these products is acetate, a chemical with various applications in industry. Metabolic engineering can be used to increase the titer of extracellular acetate in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). RESULTS: Simultaneous expression of phosphoketolase (PK) and phosphotransacetylase (Pta) resulted in an enhanced acetate titer in Synechocystis cells (Roussou et al. Metab Eng 88:250-260) [1]. In the present study these two enzymes were expressed in different locus in the genome as well as expressed in the same locus organized as a single operon. The latter design reached higher acetate production. Attempts to further optimize the production through the creation of fused protein did not result in significant higher values than 2.3 g/L previously reported. However, the production was further increased when acetate kinase (AckA) was additionally overexpressed. Cultivation of this strain in high density cultivation (CellDEG system) led to high levels of acetate with a maximum of 7.1 g/L cumulative acetate production after 12 days of experiment when the cultures were sampled every day. CONCLUSIONS: Synechocystis sp. PCC 6803 is a candidate for sustainable acetate production driven by sunlight and CO2. The high level of acetate production is result of combining genomic integration of heterogenous genes in the cell and overexpression of native genes through self-replication vector. The production level achieved through the high-density cultivation reveal the strain capabilities when the growth conditions are optimal.
This paper re-examines the definition of life, critiquing and building upon Plante's recently proposed symbiotic, holistic, and gradualist framework. Plante's model integrates symbiosis across biological scales, holism to unify hierarchical complexity, and gradualism to address the continuum between non-living and living entities. While innovative, the model omits two critical factors underpinning life: information and water. These elements form the foundation for a novel approach based on informational dissipative dynamics and Prigogine-like structures. Water is posited as a dynamic, topological medium capable of encoding and transferring information via transient hydrogen-bond networks. This phenomenon creates "informational topologies" that guide the organization of molecules, bridging the gap between physical randomness and biological order. The proposed framework explores how water properties drive the emergence of autopoietic systems through the interplay of thermodynamic, informational, and quantum dynamics. The model introduces the concept of informational entropy gradients within water-molecule interactions, facilitating the iterative development of structured, dissipative systems. These gradients sustain the system far from equilibrium, enabling life complexity and persistence. As these systems evolve, the interplay of entropic gradients, dissipative energy, and information processing leads to increased order, self-replication, and, ultimately, the emergence of life. By re-framing life as an informational dissipative process, the paper bridges gaps in Plante's approach and proposes a broader, foundational understanding of biological systems. This perspective offers a unifying framework for exploring life origins, evolution, and complexity while highlighting water's indispensable role in shaping living systems.
The integration of biological functions into a single operating system is considered a major challenge in the construction of a synthetic cell. We present autocatalytic selection (ACS) of gene functions as a driver for integrating biological modules in vitro. A gene of interest (GOI) is introduced into a minimal DNA self-replicator and the function of the GOI is linked to transcription, translation or DNA replication through a positive feedback loop. As the encoded function eventually promotes DNA self-replication, the gene variants with greater activity are selected. Using different coupling mechanisms, we demonstrate ACS of three functions: transcription, in situ regeneration of dGTP from dGMP to support DNA replication, and β-galactosidase activity. The latter example illustrates how a function that is not directly related to the Central Dogma can be selected. In addition, we show that metabolically active replicators can be enriched from a library of variants generated by random mutagenesis. This work paves the way for ACS-driven Darwinian evolution of virtually any biomolecule in vitro, streamlining the construction of increasingly complex synthetic cells as well as the engineering of biotechnologically relevant enzymes.
Before the invention of encoded protein translation, early stages of life likely relied on catalytic RNAs (ribozymes). To test how such a system could have functioned, researchers have developed ribozymes that could have provided central functions. The central function of self-replication would have required templated RNA polymerization of nucleotides, which is energetically driven in today's life forms by the use of nucleoside 5'-triphosphates (NTPs). We previously showed that ribozymes can catalyze the formation of guanosine 5'-triphosphate (GTP) from guanosine and the prebiotically plausible polyphosphorylation reagent cyclic trimetaphosphate (cTmp) by generating a guanosine triphosphorylation ribozyme (GTR) using an in vitro selection in emulsion. This ribozyme (GTR1) had a catalytic rate enhancement of about 18,000-fold but a turnover of only about 1.7. Here, we improved this ribozyme by emulsion selection from a doped library of GTR1 that was metabolically coupled to a polymerase ribozyme. High-throughput sequencing and biochemical analysis identified the most efficient variant of GTR1 with 19 mutations, which increased the GTP turnover number to ~13. Biochemical analysis of this GTR1e revealed biphasic reaction kinetics with an apparent overall KMAPP around 11 mM for cTmp. When coupled to an RNA polymerase ribozyme, up to five guanosines were incorporated into an RNA polymer, which represents an important step toward modeling an RNA-based life form in the lab.
DNA is a promising medium for next-generation data storage because of ultrahigh information density and stability. DNA storage within living organisms presents further advantages, such as self-replication, compactness, and concealment. Early efforts primarily developed predetermined methods for encoding and decoding data using in vivo DNA sequences. However, these methods may pose a security risk while opening a clear channel for potential data access and breaches. To address these challenges, we propose a unified paradigm, integrated computational-biological programming (ICBP), by exploiting the intrinsic digital characteristics within computational and microbial systems. ICBP involves the construction of dynamic code tables from gene regulatory networks or complete genomes across diverse species, expanding the key space by more than 100 orders of magnitude compared with existing methods. The encryption algorithm in ICBP benefits from DNA encoding, computing, and computational operations, leading to superior encryption quality and resistance to brute force and statistical attacks. Furthermore, we demonstrated the practical utility of ICBP via the successful encryption, microbial storage, and decryption of digital files within living systems, achieving 100% data recovery after 100 generations of replication. By combining computational logic with the biological complexities of living systems, the ICBP offers a transformative strategy for secure DNA data storage.