Nanomaterials with enzyme‐mimetic activities are possible alternatives to natural enzymes. Mimicking enzymatic enantioselectivity remains a great challenge. Herein, we report that cysteine‐derived chiral carbon dots (CDs) can mimic topoisomerase I to mediate topological rearrangement of supercoiled DNA enantioselectively. d‐CDs can more effectively catalyze the topological transition of plasmid DNA from supercoiled to nicked open‐circular configuration than l‐CDs. Experiments suggest the underlying mechanism: d‐CDs intercalatively bind with DNA double helix more strongly than l‐CDs; the intercalative CDs can catalyze the production of hydroxyl radicals to cleave phosphate backbone in one strand of the double helix, leading to topological rearrangement of supercoiled DNA. Molecular dynamics (MD) simulation show that the stronger affinity for hydrogen‐bond formation and hydrophobic interaction between d‐cysteine and DNA than that of l‐cysteine is the origin of enantioselectivity.
The design of controllable dynamic systems is vital for the construction of organelle-like architectures in living cells,b ut has proven difficult due to the lack of control over defined topological transformation of self-assembled structures.H erein, we report aD NA based dynamic assembly system that achieves lysosomal acidic microenvironment specifically inducing topological transformation from nanoparticles to organelle-like hydrogel architecture in living cells. Designer DNAn anoparticles are constructed from doublestranded DNAwith cytosine-richstick ends (C-monomer) and are internalizedi nto cells through lysosomal pathway.T he lysosomal acidic microenvironment can activate the assembly of DNAm onomers,i nducing transformation from nanoparticles to micro-sized organelle-like hydrogel which could further escape into cytoplasm. We show how the hydrogel regulates cellular behaviors:c ytoskeleton is deformed, cell tentacles are significantly shortened, and cell migration is promoted.
Cellular genes that are functionally related to each other are usually confined in specialized subcellular compartments for efficient biochemical reactions. Construction of spatially controlled biosynthetic systems will facilitate the study of biological design principles. Herein, we fabricated a gene circuit compartment by coanchoring two functionrelated genes on surface of gold nanoparticles and investigated the compartment effect on cascade gene expression in a cell-free system. The gene circuit consisted of a T7 RNA polymerase (T7 RNAP) expression cassette as regulatory gene and a fluorescent protein expression cassette as regulated reporter gene. Both the expression cassettes were attached on a Y-shaped DNA nanostructure whose other two branches were mercapto-modified in order to steadily anchor the gene expression cassettes on the surface of gold nanoparticles. Experimental results demonstrated that both the yield and initial expression rate of the fluorescent reporter protein in the gene circuit compartment system were enhanced compared with those in free gene circuit system. Mechanism investigation revealed that the gene circuit compartment on nanoparticle made the regulatory gene and regulated reporter gene spatially proximal at nanoscale, thus effectively improving the transfer efficiency of the regulatory proteins (T7 RNAP) from regulatory genes to the regulated reporter genes in the compartments, and consequently, the biochemical reaction efficiency was significantly increased. This work not only provided a simplified model for rational molecular programming of genes circuit compartments on nanointerface but also presented implications for the cellular structure−function relationship.
Nanomaterials with enzyme‐mimetic activities are possible alternatives to natural enzymes. Mimicking enzymatic enantioselectivity remains a great challenge. Herein, we report that cysteine‐derived chiral carbon dots (CDs) can mimic topoisomerase I to mediate topological rearrangement of supercoiled DNA enantioselectively. d‐CDs can more effectively catalyze the topological transition of plasmid DNA from supercoiled to nicked open‐circular configuration than l‐CDs. Experiments suggest the underlying mechanism: d‐CDs intercalatively bind with DNA double helix more strongly than l‐CDs; the intercalative CDs can catalyze the production of hydroxyl radicals to cleave phosphate backbone in one strand of the double helix, leading to topological rearrangement of supercoiled DNA. Molecular dynamics (MD) simulation show that the stronger affinity for hydrogen‐bond formation and hydrophobic interaction between d‐cysteine and DNA than that of l‐cysteine is the origin of enantioselectivity.
The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as “gene compartments” for cell‐free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane‐free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of “cellular” physical environments on biochemical reactions in simplified cell‐free systems.
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