Ribozyme activity modulates the physical properties of RNA-peptide coacervates.

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

Artificial cell design: reconstructing biology for life science applications.

Artificial cells are developed to redesign novel biological functions in a programmable and tunable manner. Although it aims to reconstitute living cell features and address 'origin of life' related questions, rapid development over the years has transformed artificial cells into an engineering tool with huge potential in applied biotechnology. Although the application of artificial cells was introduced decades ago as drug carriers, applications in other sectors are relatively new and could become possible with the technological advancement that can modulate its designing principles. Artificial cells are non-living system that includes no prerequisite designing modules for their formation and therefore allow freedom of assembling desired biological machinery within a physical boundary devoid of complex contemporary living-cell counterparts. As stimuli-responsive biomimetic tools, artificial cells are programmed to sense the surrounding, recognise their target, activate its function and perform the defined task. With the advantage of their customised design, artificial cells are being studied in biosensing, drug delivery, anti-cancer therapeutics or artificial photosynthesis type fields. This mini-review highlights those advanced fields where artificial cells with a minimalistic setup are developed as user-defined custom-made microreactors, targeting to reshape our future 'life'.

Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.

The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.

Constrained α-Helical Peptides as Inhibitors of Protein-Protein and Protein-DNA Interactions.

Intracellular regulatory pathways are replete with protein-protein and protein-DNA interactions, offering attractive targets for therapeutic interventions. So far, most drugs are targeted toward enzymes and extracellular receptors. Protein-protein and protein-DNA interactions have long been considered as "undruggable". Protein-DNA interactions, in particular, present a difficult challenge due to the repetitive nature of the B-DNA. Recent studies have provided several breakthroughs; however, a design methodology for these classes of inhibitors is still at its infancy. A dominant motif of these macromolecular interactions is an α-helix, raising possibilities that an appropriate conformationally-constrained α-helical peptide may specifically disrupt these interactions. Several methods for conformationally constraining peptides to the α-helical conformation have been developed, including stapling, covalent surrogates of hydrogen bonds and incorporation of unnatural amino acids that restrict the conformational space of the peptide. We will discuss these methods and several case studies where constrained α-helices have been used as building blocks for appropriate molecules. Unlike small molecules, the delivery of these short peptides to their targets is not straightforward as they may possess unfavorable cell penetration and ADME properties. Several methods have been developed in recent times to overcome some of these problems. We will discuss these issues and the prospects of this class of molecules as drugs.

A Potent Conformation-Constrained Synthetic Peptide Mimic of a Homeodomain Selectively Regulates Target Genes in Cells.

DNA, as a target for therapeutic intervention, remains largely unexplored. DLX-4, a homeodomain containing transcription factor, and its spliced isoforms play crucial roles in many aspects of cellular biochemistry and important roles in many diseases. A smaller peptide mimicking the homeodomain of the transcription factor DLX-4 was designed and synthesized by suitable conjoining of its modified DNA-binding elements. The peptide binds to DLX-4 target sites on the regulatory region of the globin gene cluster with native-like affinity and specificity in vitro. When conjugated to cell penetrating and nuclear localization sequences, it upregulated some of the genes repressed by DLX-4 or its isoforms, such as ß- and γ-globin genes in erythropoietin-induced differentiating CD34+ human hematopoietic stem/progenitor cells with high specificity by competing with the respective binding sites. Engineered peptides mimicking DNA-binding domains of transcription factors offer the potential for creating synthetic molecules for directly targeting DNA sites with high specificity.

A peptide-based synthetic transcription factor selectively activates transcription in a mammalian cell.

A peptide-based cell permeable synthetic transcription factor is reported, which binds to its target site with high affinity and specificity. When linked to a HAT-binding peptide, it causes significant upregulation of gene expression in a mammalian cell. Such molecules may be developed for selectively activating repressed genes in mammalian cells.

Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences.

The nature of the interface of specific protein-DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3-Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DNA.