Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity.
Ontology highlight
ABSTRACT:
SUBMITTER: Wan X
PROVIDER: S-EPMC7686491 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Lung cancer is the most common cancer in the world and several miRNAs are associated with it. MiRNA sponges are presented as tools to inhibit miRNAs. We designed a system to capture miRNAs based on circular RNAs (circRNA). To demonstrate its usefulness, we chose miR-21, which is upregulated and implicated in lung cancer. We constructed a miR-21 sponge and inserted it into a vector that facilitates circular RNA production (Circ-21) to study its effect on growth, colony formation, and migration in lung cancer cell lines and multicellular tumor spheroids (MTS). Circ-21 induced a significant and time-dependent decrease in the growth of A549 and LL2 cells, but not in L132 cells. Furthermore, A549 and LL2 cells transfected with Circ-21 showed a lower number of colonies and migration than L132. Similar findings were seen in A549 and LL2 Circ-21 MTS, which showed a significant decrease in volume growth, but not in L132 Circ-21 MTS. Based on this, the miR-21 circular sponge may suppress the processes of tumorigenesis and progression. Therefore, our system based on circular sponges seems to be effective, as a tool for the capture of other miRNAs.
| S-EPMC8955967 | biostudies-literature
Project description:A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.
| S-EPMC4731013 | biostudies-literature
Project description:Microbial engineering often requires fine control over protein expression--for example, to connect genetic circuits or control flux through a metabolic pathway. To circumvent the need for trial and error optimization, we developed a predictive method for designing synthetic ribosome binding sites, enabling a rational control over the protein expression level. Experimental validation of >100 predictions in Escherichia coli showed that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicted that reusing identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing protein expression to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach should accelerate the construction and systematic optimization of large genetic systems.
| S-EPMC2782888 | biostudies-literature
Project description:One commonly performed bioinformatics task is to infer functional regulation of transcription factors by observing differential expression under a knockout, and integrating DNA binding information of that transcription factor. However, until now, this task has required dedicated bioinformatics support to perform the necessary data integration. GenomeSpace provides a protocol, or "recipe", and a user interface with inter-operating software tools to identify protein occupancies along the genome from a ChIP-seq experiment and associated differentially regulated genes from a RNA-Seq experiment. By integrating RNA-Seq and ChIP-seq analyses, a user is easily able to associate differing expression phenotypes with changing epigenetic landscapes.
| S-EPMC5806050 | biostudies-literature
Project description:Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.
| S-EPMC7414067 | biostudies-literature
Project description:BackgroundSingle-cell DNA sequencing is getting indispensable in the study of cell-specific cancer genomics. The performance of computational tools that tackle single-cell genome aberrations may be nevertheless undervalued or overvalued, owing to the insufficient size of benchmarking data. In silicon simulation is a cost-effective approach to generate as many single-cell genomes as possible in a controlled manner to make reliable and valid benchmarking.ResultsThis study proposes a new tool, SCSilicon, which efficiently generates single-cell in silicon DNA reads with minimum manual intervention. SCSilicon automatically creates a set of genomic aberrations, including SNP, SNV, Indel, and CNV. Besides, SCSilicon yields the ground truth of CNV segmentation breakpoints and subclone cell labels. We have manually inspected a series of synthetic variations. We conducted a sanity check of the start-of-the-art single-cell CNV callers and found SCYN was the most robust one.ConclusionsSCSilicon is a user-friendly software package for users to develop and benchmark single-cell CNV callers. Source code of SCSilicon is available at https://github.com/xikanfeng2/SCSilicon .
| S-EPMC9092674 | biostudies-literature
Project description:Limited sample sizes can lead to spurious modeling findings in biomedical research. The objective of this work is to present a new method to generate synthetic populations (SPs) from limited samples using matched case-control data (n = 180 pairs), considered as two separate limited samples. SPs were generated with multivariate kernel density estimations (KDEs) with unconstrained bandwidth matrices. We included four continuous variables and one categorical variable for each individual. Bandwidth matrices were determined with Differential Evolution (DE) optimization by covariance comparisons. Four synthetic samples (n = 180) were derived from their respective SPs. Similarity between observed samples with synthetic samples was compared assuming their empirical probability density functions (EPDFs) were similar. EPDFs were compared with the maximum mean discrepancy (MMD) test statistic based on the Kernel Two-Sample Test. To evaluate similarity within a modeling context, EPDFs derived from the Principal Component Analysis (PCA) scores and residuals were summarized with the distance to the model in X-space (DModX) as additional comparisons. Four SPs were generated from each sample. The probability of selecting a replicate when randomly constructing synthetic samples (n = 180) was infinitesimally small. MMD tests indicated that the observed sample EPDFs were similar to the respective synthetic EPDFs. For the samples, PCA scores and residuals did not deviate significantly when compared with their respective synthetic samples. The feasibility of this approach was demonstrated by producing synthetic data at the individual level, statistically similar to the observed samples. The methodology coupled KDE with DE optimization and deployed novel similarity metrics derived from PCA. This approach could be used to generate larger-sized synthetic samples. To develop this approach into a research tool for data exploration purposes, additional evaluation with increased dimensionality is required. Moreover, given a fully specified population, the degree to which individuals can be discarded while synthesizing the respective population accurately will be investigated. When these objectives are addressed, comparisons with other techniques such as bootstrapping will be required for a complete evaluation.
| S-EPMC7839232 | biostudies-literature
Project description:The application of stem cell-derived secretome in regenerative therapies offers the key advantage that instead of the stem cells, only their effective paracrine compounds are in vivo delivered. Ideally, the secretome can be steered by the culture conditions of the stem cells. So far, most studies use stem cells cultured on stiff plastic substrates, not representative of their native 3D environment. In this study, cells are cultured inside synthetic polyisocyanide (PIC)-based hydrogels, which are minimal, tailorable, and highly reproducible biomimetic matrices. Secretome analysis of human adipose-derived stem cells (multiplex, ELISA) displays that matrix manipulation is a powerful tool to direct the secretome composition. As an example, cells in nonadherent PIC gels secrete increased levels of IL-10 and the conditioned media from 3D culture accelerate wound closure. In all, our PIC-based approach opens the door to dedicated matrix design to engineer the secretome for custom applications.
| S-EPMC7760093 | biostudies-literature
Project description:Antimicrobial peptides have attracted considerable interest as potential new class of antibiotics against multi-drug resistant bacteria. However, their therapeutic potential is limited, in part due to susceptibility towards enzymatic degradation and low bioavailability. Peptoids (oligomers of N-substituted glycines) demonstrate proteolytic stability and better bioavailability than corresponding peptides while in many cases retaining antibacterial activity. In this study, we synthesized a library of 36 peptoids containing fluorine, chlorine, bromine and iodine atoms, which vary by length and level of halogen substitution in position 4 of the phenyl rings. As we observed a clear correlation between halogenation of an inactive model peptoid and its increased antimicrobial activity, we designed chlorinated and brominated analogues of a known peptoid and its shorter counterpart. Short brominated analogues displayed up to 32-fold increase of the activity against S. aureus and 16- to 64-fold against E. coli and P. aeruginosa alongside reduced cytotoxicity. The biological effect of halogens seems to be linked to the relative hydrophobicity and self-assembly properties of the compounds. By small angle X-ray scattering (SAXS) we have demontrated how the self-assembled structures are dependent on the size of the halogen, degree of substitution and length of the peptoid, and correlated these features to their activity.
| S-EPMC7481290 | biostudies-literature
Project description:Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.
| S-EPMC5007465 | biostudies-literature