Schizokinen and its analogues - MS2 - molecular networking
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ABSTRACT: MS2 experiments with 3 CE: 10, 20, 30 eV for schizokinen derivatives, schizokinen, schizokinen A, N-deoxy-schizokinen and N-deoxy-schizokinen A
Project description:MS2 experiments with 3 CE: 10, 20, 30 eV for schizokinen derivatives, schizokinen, schizokinen A, N-deoxy-schizokinen and N-deoxy-schizokinen A
Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RybB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RybB in a rne131 ΔrybB strain. RybB (without MS2) was used as control
Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RyhB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RyhB in a rne131 ?ryhB strain. RyhB (without MS2) was used as control
Project description:MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaG sRNA targetome. Affinity purification of in vivo regulatory complexes coupled with high throughput RNA sequencing methodology or MAPS standing for “MS2 affinity purification coupled to RNA".
Project description:Since the introduction of the online open-source GNPS, molecular networking has quickly become a widely applied tool in the field of natural products chemistry, with applications from dereplication, genome mining, metabolomics, and visualization of chemical space. Studies have shown that data dependent acquisition (DDA) parameters affect molecular network topology but are limited in the number of parameters studied. With an aim to optimize LC-MS2 parameters for integrating GNPS-based molecular networking into our marine natural products workflow, a design of experiment (DOE) was used to screen the significance of the effect that eleven parameters have on both Classical Molecular Networking workflow (CLMN) and the new Feature-Based Molecular Networking workflow (FBMN). Our results indicate that four parameters (concentration, run duration, collision energy and number of precursors per cycle) are the most significant data acquisition parameters affecting the network topology. While concentration and the LC duration were found to be the two most important factors to optimize for CLMN, the number of precursors per cycle and collision energy were also very important factors to optimize for FBMN.
Project description:RNA delivery is a method of choice to achieve transient gene expression for research and in cell- or gene-based therapies. To improve retroviral transfer, we designed a dimerization-independent MS2-driven packaging system using MS2-retrovirus chimeras. We delivered RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem-cells. We used microarrays to detail the global programme of gene expression confirming the effects of pro-osteogenic genes transduced by MS2 chimeric lentiviral particles.
Project description:The Myc/Max heterodimer has crucial roles in normal cellular processes such as cell proliferation, metabolism, apoptosis, and differentiation, but its activity is often deregulated in a majority of human cancers. In an effort to explore alternative modes of Myc perturbation, we identified KI-MS2-008 as a small molecule that binds Max and modulates Myc-driven transcription, and in some cellular contexts, KI-MS2-008 treatment leads to a decrease in c-Myc protein levels. As the Myc/Max heterodimer controls many cellular processes, we expected that treatment with this small molecule would cause changes in the transcriptome. We found that treatment with 10 µM KI-MS2-008 resulted in global alterations in the transcriptome, mimicking direct Myc inactivation with doxycycline in P493-6, a B cell line with a Tet-Off system for c-Myc expression. We also discovered enrichment of various Myc target gene sets in the genes downregulated in response to KI-MS2-008 treatment in P493-6 cells. This trend was also observed in ST486 cells, but not in P3HR1 cells, which were chosen as non-engineered B cell lines that were sensitive and insensitive, respectively, toward KI-MS2-008 in cell viability assays.
Project description:The samples were analyzed by Dr. Ernest Oppong-Danquah, while molecular newtorking and chemoinformatic analysis were operated by Dr. Giovanni Andrea Vitale. The sample runs were executed in positive mode with the following conditions: capillary voltage: 3.0 kV, sample cone voltage: 30 V, source temperature: 150 C. A scan range from 50 to 1200 Da was used, MS2 fragmentation was achieved in DDA mode, with ramp collision energy: Low CE from 20_60 eV and a high CE of 40_80 eV.
Project description:During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. The 3’ETS of the glyW-cysT-leuZ polycistronic tRNA precursor was highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs were shown to base pair with the same region in the 3’ETS of leuZ (3’ETSleuZ). Disrupting the pairing by mutating 3’ETSleuZ significantly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3’ETSleuZ prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and increases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3’ETSleuZ functions as a sponge to absorb transcriptional noise from repressed sRNAs. Finally, the fact that RybB and MicF sRNAs are co-purified with ITSmetZ-metW and ITSmetW-metV strongly suggests a much broader phenomenon. Identification of sRNAs co-purified with MS2-ITSmetZW and MS2-ITSmetWV. ITSmetZW and ITSmetWV (without MS2) were used as control