5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), first method for quantitative mapping of 5hmC genomic at single-nucleotide resolution. Selective chemical oxidation 5-formylcytosine (5fC) enables conversion 5fC uracil. demonstrate utility oxBS-Seq map and quantify CpG islands (CGIs) mouse embryonic stem (ES) cells identify 800 5hmC-containing CGIs that...

DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) DNA. These derivatives a role demethylation but addition may signaling functions their own right. A recent study identified proteins which showed preferential binding 5-methylcytosine its oxidised forms, where readers for 5hmC little overlap, bound further oxidation...

Methylation of cytosine in DNA (5mC) is an important epigenetic mark that involved the regulation genome function. During early embryonic development mammals, methylation landscape dynamically reprogrammed part through active demethylation. Recent advances have identified key players demethylation pathways, including oxidation 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by TET enzymes, excision 5fC base repair enzyme thymine glycosylase (TDG). Here, we provide first...

We have previously used three-dimensional (3D) printing to prepare tissue-like materials in which picoliter aqueous compartments are separated by lipid bilayers. These printed droplets elaborated into synthetic cells using a tightly regulated vitro transcription/translation system. A light-activated DNA promoter has been developed that can be turn on the expression of any gene within cells. light activation express protein pores 3D-printed patterns tissues. The incorporated specific bilayer...

Biological cells display complex internal architectures with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts create advanced cell mimics, namely, artificial cells, demands strategies for constructing similarly heterogeneous structures localized functionalities. Here, we introduce a platform membraneless from self-assembly of synthetic DNA nanostructures in which domains can be established thanks prescribed...

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ expression and, thereby, cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within was using light by encoding genes of interest on light-activated templates. Light-activated contained photocleavable...

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications biotechnology. Developing methods control DNA templates for cell-free will be important precise regulation of complex pathways use with synthetic cells, particularly using remote, nondamaging stimuli such as visible light. Here, we have synthesized blue light-activatable parts that tightly regulate RNA protein synthesis. We found this light-activated...

Within many chemical and biological systems, both synthetic natural, communication via messengers is widely viewed as a key feature. Often known molecular communication, such has been concern in the fields of biologists, nanotechnologists, communications engineers, philosophers science. However, interactions between these are currently limited. Nevertheless, fact that same basic phenomenon studied by all raises question whether there unexploited interdisciplinary synergies. In this paper, we...

Synthetic tissues can be generated by forming networks of aqueous droplets in lipid-containing oil. Each droplet contains a cell-free expression system and is connected to its neighbor through lipid bilayer. In the present work, we have demonstrated precise external control such activating protein within single droplets, using light-activated DNA encode either fluorescent or pore-forming protein. By controlling extent activation, synthetic were with graded levels patterns droplets. Further,...

Cell-free expression of a gene to protein has become vital tool in nanotechnology and synthetic biology. Remote-control cell-free systems with multiple, orthogonal wavelengths light would enable precise, noninvasive modulation, opening many new applications biology medicine. While there been success developing ON switches, the development OFF switches lacking. Here, we have developed orthogonally light-controlled by attaching nitrobenzyl coumarin photocages antisense oligonucleotides. These...

Antisense oligonucleotides (ASOs) are a promising class of therapeutics designed to modulate gene expression. Both key mechanisms action for ASOs operate in the nucleus: splice-switching modify pre-mRNA, processed nucleus, and mRNA-degrading require RNase H, an enzyme predominantly active nucleus. Therefore, achieve maximal efficacy, efficient nuclear delivery. Current ASO shuttle out nucleus inefficiently. In this work, we have synthesised conjugates import, by covalent conjugation with...

Antisense oligonucleotides (ASOs) can modulate gene expression at the mRNA level, providing ability to tackle conventionally undruggable targets and usher in an era of personalized medicine. A key mode action for ASOs relies upon RNase H-engagement nucleus, however, site mature is cytoplasm. This disconnect limits efficacy biomedical applications ASOs. In this paper, we have established a new mechanism achieving potent targeted knockdown by leveraging lysosomal degradation pathway. To...

Targeted protein degradation is a powerful therapeutic approach: expanding the druggable proteome, providing enhanced selectivity, and having ability to overcome conventional resistance mechanisms. A major class of such molecules are proteolysis-targeting chimeras (PROTACs). PROTACs catalytic heterobifunctional small that simultaneously bind interest (POI) an E3 ligase. These induce proximity-dependant ubiquitination POI, which causes its subsequent by ubiquitin–proteasome system. While have...

Synthetic cells, like their biological counterparts, require internal compartments with distinct chemical and physical properties where different functionalities can be localized. Inspired by membrane-less compartmentalization in here, we demonstrate how microphase separation used to engineer heterogeneous cell-like architectures programmable morphology compartment-targeted activity. The synthetic cells self-assemble from amphiphilic DNA nanostructures, producing core-shell condensates due...

Abstract Synthetic tissues built from communicating aqueous droplets offer potential applications in biotechnology, however, controlled release of their contents has not been achieved. Here we construct two-droplet synthetic tissue modules that function an environment. One droplet contains a cell-free protein synthesis system and prodrug-activating enzyme the other small-molecule prodrug analog. When Zn 2+ -sensitive pore is made first droplet, it allows to migrate second become activated by...

A key goal of bottom-up synthetic biology is to construct cell- and tissue-like structures. Underpinning cellular life the ability process several external chemical signals, often in parallel. Until now, structures have been constructed with no more than one signaling pathway. Many pathways rely on signal transport across membranes using protein nanopores. However, such systems currently suffer from slow molecules. We optimized application these nanopores permit fast molecular transport,...

Abstract Controlling cell-free expression of a gene to protein with non-invasive stimuli is vital the future application DNA nanodevices and synthetic cells. However, little emphasis has been placed on developing light-controlled ‘off’ switches for expression. Light-activated antisense oligonucleotides have developed induce knockdown in living cells; however, they are complicated synthesise not tested systems. Developing simple, accessible methods produce light-activated will be crucial...

Quenched chiral perturbation theory is extended to include heavy-light mesons. Nonanalytic corrections the decay constants, Isgur-Wise function, and masses mixing of heavy mesons are then computed. The results used estimate error due quenching in lattice computations these quantities. For reasonable choices parameters, it found that has a strong effect on ${\mathit{f}}_{\mathit{B}\mathit{s}}$/${\mathit{f}}_{\mathit{B}}$, reducing by as much 28%. errors essentially negligible for function parameter.