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opencc-by-4.0Sep 2024View details →

Linked multipoint cotranscriptional RNA chemical probing data for the E. coli SRP RNA, C. beijerinckii pfl ZTP riboswitch, and B. cereus crcB fluoride riboswitch

Cotranscriptional RNA chemical probing data for the E. coli signal recognition particle (SRP) RNA, Clostridium beijerinckii pfl ZTP riboswitch, and Bacillus cereus crcB fluoride riboswitch. Data were generated using the linked multipoint Transcription Elongation Complex RNA structure probing (TECprobe-LM) and variable length Transcription Elongation Complex RNA structure probing (TECprobe-VL) procedures.

opencc-by-4.0Jun 2021View details →

Are Enantiomer of Chemical Probes Good Negative Control?

Chemical probes are drug-like compounds that bind potently and selectively to a protein target. When there is a chiral center in the compound, a common practice is to purify and test both enantiomers. Sometimes (often) enantiomer A is active and B is inactive or far less potent. When this is the case, enantiomer A is declared the chemical probe, while enantiomer B is used as a negative control . But if enantiomer A is active against unknown off-targets, enantiomer B may be inactive against these off-targets, which would be a serious problem. The project is to systematically evaluate how often enantiomer B is inactive against protein X if enantiomer A is active against the same target.

opencc-by-4.0Aug 2021View details →

Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe

Dataset details Project Title: Chemoproteomic Analysis of an NSD2-PWWP1 Chemical Probe Keywords: chemoproteomics, epigenetics, chemical probe, histone methyltransferase Project description: Here we use competitive chemoproteomics pulldowns followed by label-free quantitative LC-MS/MS to assess target engagement and selectivity profiles of UNC6934 and UNC7145, a chemical probe targeting the PWWP1 domain of NSD2 and its negative control counterpart, respectively. To this end, we used a biotinylated probe derivative (UNC7096) for streptavidin pulldowns from KMS-11 multiple myeloma cell lysates, including in the context of UNC7145 or UNC6934 competition. Methods: Chemical Proteomics  To prepare whole cell lysates, KMS11 cells were washed 2 times with 1x PBS, lysed by resuspension in high-salt lysis buffer (20 mM HEPES pH 7.5, 350 mM KCl, 1% Triton X-100 + a protease inhibitor cocktail containing aprotinin, leupeptin, pepstatin A, and E-64) and passed through a 25 gauge needle 5 times followed by a 20 min incubation on ice. Cell lysates were cleared by centrifugation at 18 000 x g for 20 minutes at 4°C. Cleared supernatant was diluted to 150 mM KCl and 0.4% Triton X-100 with 20mM HEPES pH7.5 including fresh protease inhibitors. Sample protein concentrations were determined using the BCA assay (ThermoScientific). For each pulldown, 3 mg of cell lysate was pre-incubated with either DMSO control, 20 µM UNC7145, or 20 µM UNC6934 (final concentration) for 1 hour with rotation at 4°C. For each sample, 25 µl of M270 Dynabeads (ThermoScientific) were prepared by washing three times in low salt wash buffer (10 mM Tris-HCl pH7.9, 100 mM NaCl, 0.1% NP-40), followed by incubation with 1 µM UNC7096 (biotinylated probe) for 1 hour at 4 °C. The unbound biotinylated compound was removed by 3 washes with low salt buffer. UNC7096 bound beads were then added to each sample followed by incubation 1 hour with rotation at 4oC. Beads were then washed 3 times with low-salt wash buffer followed by 2 washes with 50mM ammonium bicarbonate. On-bead digestion was performed by overnight incubation at 37°C with 2 µg of mass spectrometry grade trypsin (Promega). The following morning an additional 2 µg of trypsin was added to each sample and incubated at 37°C for 4-6 hours. The supernatant, containing digested peptides, was collected. Beads were then washed twice with water and supernatant pooled with digested peptides. Samples were then acidified with formic acid to a final concentration of 2% final concentration and flash frozen prior drying under vacuum before being run on a Thermo Scientific LTQ Orbitrap Velos. Label-free quantitative mass spectrometry data analysis  Raw MS/MS files were searched and quantified using Maxquant version 1.6.7.0 using the UP000005640 Uniprot human database (containing 20,605 protein entries, last modified November 5, 2019) with label-free quantification enabled and variable modifications oxidized methionine (+15.9949 Da) and deamidated asparagine (+0.9840) set. First search peptide tolerance and main search peptide tolerance were set at 30 and 6 ppm, respectively. For all other parameters default settings were used.  Differential enrichment analysis was performed using the DEP package (v1.8.0) in R (v3.5.1). Briefly, samples were filtered for proteins identified in 2 out of 3 replicates of at least one condition, normalized by variance stabilizing normalization and tested for differential enrichment relative to pulldowns competed with DMSO vehicle control.  Brief Description of Data Files: <ul> <li>.raw & .index files - raw proteomic data files - note: dataset has also been uploaded to ProteomeXchange Consortium via the PRIDE17 partner repository with the dataset identifier PXD017641. </li> <li>mqpar.xml - provides parameters used for quantification with Maxquant, which can be found in the combined folder. </li> <li>UP000005640_9606.fasta - UniProt reference used for Mazquant quantification of peptides</li> <li>NSD2_Chemoproteomics.Rproj & DEP_Analysis.R - R project & script file for differential analysis of Maxquant output with DEP. </li> <li>methods.docx - additional details covering experimental method</li> </ul>

opencc-by-4.0Sep 2021View details →

Chemical probing of U2 snRNA - Capillary electrophoresis chromatograms

1m7 chemical probing of the synthetic RNA consisting of the first 100 nucleotides of U2 snRNA along with a series of mutations in the Stem I region and data analysis was carried out essentially as described previously in "Kladwang W, VanLang CC, Cordero P, Das R (2011) A two-dimensional mutate-and-map strategy for non-coding RNA structure. Nat Chem 3: 954-962".  Briefly synthetic U2 snRNA templates flanked by two stable stem-loops to use as probing standards and a 3′ tail for primer extensions were generated by PCR from overlapping oligonucleotides and used in T7 run-off transcription reactions. The transcribed RNA was separated on a 5% (v/v) denaturing polyacrylamide gel. Correlating bands identified by UV shadowing were excised, and the RNA was isolated from excised bands by soaking gel slices in 0.3 M sodium acetate pH 4.8, 1 mM EDTA, 10% phenol overnight followed by ethanol precipitation. For chemical probing, 0.6 pmol of RNA in 50mM Na-HEPES was heated to 95 ˚C for 3 minutes and allowed to fold at room temperature for 20 minutes followed by the addition of 10 mM magnesium chloride. The RNA was aliquoted into a 96-well plate with 5 mM 1m7 or water and incubated for 10 minutes at room temperature followed by the addition of oligo dT magnetic beads (Ambion) in 0.25 M Na-MES pH 6.0, 1.5 M NaCl, and 6.4 nM FAM-labeled primer. RNA/primers were isolated by magnetic bead immobilization, washed twice with 70% ethanol, and resuspended in 2.5 μL water. For reverse transcription, 20 units SuperScript III reverse transcriptase (Thermo Fisher Scientific), 5 mM dithiothreitol, 0.8 mM dNTPs, 50 mM Tris-HCl pH 8.3, 75 mM potassium chloride, and 3 mM magnesium chloride was added, and reactions were incubated for 30 minutes at 48˚C. Ladders were generated by the addition of ddNTPs to additional control reactions. Following reverse transcription, 0.2 M sodium hydroxide was added, and samples were incubated for 3 minutes at 90˚C to degrade the remaining RNA. Samples were neutralized by the addition of 1.4 M sodium chloride and 0.6 M hydrogen chloride, followed by 1.3 M sodium acetate pH 5.2. DNA was purified by magnetic bead immobilization, washed twice with 70% ethanol, and resuspended in Hi-Di formamide supplemented with R0X350 dye standards (1:8 ROXF) (Thermo Fisher Scientific). 1:3 and 1:15 dilutions of the samples in ROXF were analyzed by capillary electrophoresis (ELIM Biopharmaceuticals).

opencc-by-4.0Feb 2023View details →

Cotranscriptional RNA chemical probing data for ZTP, fluoride, and ppGpp riboswitches

Cotranscriptional RNA chemical probing data for the Clostridium beijerinckii pfl ZTP riboswitch, Bacillus cereus crcB fluoride riboswitch, and Clostridiales bacterium oral taxon 876 str. F0540 ppGpp riboswitch. Data were generated using the Transcription Elongation Complex RNA structure probing-multilength (TECprobe-ML) and single length (TECprobe-SL) procedures.

opencc-by-4.0Aug 2024View details →

Nanoscale probing of the organic binder in artists' paint layers: organic phases and chemical dynamics - Dataset

Open the record for dataset details and reuse information.

opencc-by-4.0Oct 2024View details →

Investigating the interplay between RNA structural dynamics and RNA chemical probing experiments

Normalized SHAPE reactivity for pre-miR20b and CDE2GG as reported in 'Investigating the interplay between RNA structural dynamics and RNA chemical probing experiments'.

Fig. 5. A in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 5. A Reactivity of the α-alectoronic acid 18 and the α-collatolic acid 19 with K. B Structure of the perlatolic acid 20.

Fig. 2 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 2. δ-keto ester-containing depsides considered in this study. 1 (Elix and Whitton, 1989); 2 (Elix et al., 1996; Elix and Wardlaw, 1997; Orange, 2014); 3 (Culberson et al., 1977; P´erez et al., 2016); 4 (Huneck et al., 1970).

Fig. 9 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 9. KUV probe on extracts of P. cinereoatra C, P. speirea var speirea S, P. irriguaI I, P. macrocarpa Ma and P. platycarpoides P. Extracts were spotted and K was added (right) or not (left).

Fig. 1 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 1. Molecular structures of glomelliferic acid 1 and glomellic acid 2 and their fluorescent behaviour upon addition of K. Key 2-oxoalkyl motif highlighted in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10. A in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 10. A. Direct application of the KUV probe on the medulla of P. cinereoatra; B. KUV probe on the eluted TLC (solvent G) of Porpidia cinereoatra C, P. tuberculosa T, P. speirea var speirea S, P. melinodes M, P. contraponenda Co, P. irrigua I and P. rugosa R.

Fig. 8 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 8. Relaxed coordinate scans of simplified despides under A. neutral and B. basic conditions; C. Energy minimized structure of simplified depsidone under basic conditions; D. Manually adjusted dihedral angle to position the enolate close to the ester carbonyl group.

Fig. 11 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 11. Structural data of compound s8 (NMR in acetone-d6).

Fig. 4 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 4. Reactivity of the methyl, 2′-O-methylmicrophyllinate 4′ with K.

Fig. 3 in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 3. Reactivity of depsides with K.

Fig. 7. A. Depsides rearrangement mechanisms with K in Intramolecular transesterification of depsides yields fluorescent 1H-isochromen-1-ones: Application as a chemical probe for lichen determination

Fig. 7. A. Depsides rearrangement mechanisms with K; B. MS fragmentation.