Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Comparative Analysis of Alkyne- and Desthiobiotinylated Photoaffinity Probes for Chemotranscriptomic Profiling

ABSTRACT: Understanding small molecule-RNA interactions is a crucial part in drug development and fundamental biology. Chemotranscriptomic profiling is emerging as a powerful platform to interrogate interactions of small molecules with entire transcriptomes. This technique relies on photoaffinity probes that covalently capture small molecule RNA interactions. Most photoaffinity probes bear an alkyne handle that requires additional inefficient functionalization and purification steps after RNA capture. We sought to improve the workflow by directly desthiobiotinylating a photoaffinity probe, omitting these additional alkyne functionalization steps. Here, we compare the suitability of desthiobiotin and alkyne modified Ribocil-derived photoaffinity probes for chemotranscriptomic profiling. Our results demonstrate binding of both photoaffinity probes to their specific target, the FMN riboswitch, using in vitro transcription/translation and RT-qPCR. We also observed high unspecific interactions due to proposed weak and nonspecific binding of the desthiobiotin moiety to RNA analyzed by dot blots and RT-qPCR. Finally, transcriptome-wide sequencing confirmed the unselective interaction of desthiobiotin. These findings suggest that desthiobiotin is an inefficient enrichment handle for the design of photoaffinity probes, resulting in many off-target interactions.

ORGANISM(S): Escherichia coli

PROVIDER: GSE318057 | GEO | 2026/03/18

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Design and evaluation of a cyclobutane diazirine alkyne tag for photoaffinity labeling in cells

Project description:Alkyl diazirines are frequently used in photoaffinity labeling to trap and identify small molecule–protein interactions. How-ever, the alkyl diazirine can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a long-lived alkyl diazo intermediate. To alter this reactivity preference, we developed PALBOX, a novel cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity. We show that PALBOX is readily derivatized and attached to small molecules to profile their binding interactions in cells. Systematic experiments with biological sub-strates show that the cyclobutane diazirine scaffold does not exhibit reactivity characteristic of unconstrained alkyl diazo in-termediates. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label known protein targets in cells with higher specificity. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule–protein binding sites. Thus, PALBOX is a versatile diazirine-based pho-toaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule–protein interactions.

2024-01-04 | PXD035954 | Pride

pChem_benchmarking_dataset_redoxome

Project description:Data sets are used for benchmarking, pChem, a modification-centric, blind-search tool to provide a streamlined pipeline for unbiased assessing of the performance of chemoproteomic probes. Data were produced from the redoxome profiling experiments using oxoform-specific probes as indicated, including SOH probes (DYn-2, BTD, WYneC/O/N) and SO2H probe (DiaAlk). All these probes contain a clickable alkyne handle.

2023-02-27 | PXD027764 | Pride

pChem_benchmarking_dataset_lysinome_profiling

Project description:Data sets are used for benchmarking, pChem, a modification-centric, blind-search tool to provide a streamlined pipeline for unbiased assessing of the performance of chemoproteomic probes. Data were produced from the IPM-based QTRP (quantitative thiol reactivity profiling) applications in various species, incData sets are used for benchmarking, pChem, a modification-centric, blind-search tool to provide a streamlined pipeline for unbiased assessing of the performance of chemoproteomic probes. Data were produced from the lysinome profiling experiments using amine-reactive probes as indicated, including STP and NHS, both with a clickable alkyne handle.

2022-12-16 | PXD027789 | Pride

Activity-based protein profiling in human M2 macrophages using DHA-based photoaffinity probes

Project description:17-Hydroxydocosahexaenoic acid (17-HDHA), an oxidative metabolite of the fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3), is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of 17-HDHA are poorly understood. Here, we report the design, synthesis and application of a complementary pair of bioorthogonal photoreactive probes based on the polyunsaturated scaffold of 17-HDHA and DHA. In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific protein interaction partners of 17-HDHA in primary human macrophages, which led to the identification of prostaglandin reductase 1 (PTGR1) as a target of 17-HDHA, but not DHA. Ensuing biochemical studies revealed that PTGR1 converted 17-HDHA into 17-oxo-DHA, which inhibited the biosynthesis of the pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils.

2023-04-28 | PXD027600 | Pride

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

Project description:RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enables the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.

2020-08-17 | GSE156312 | GEO

A selective photoaffinity probe enables assessment of cannabinoid CB2 receptor expression and ligand engagement in human cells

Project description:Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photo-click chemistry and hold promise as biomarkers in translational drug discovery.

2018-06-15 | PXD008460 | Pride

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

Project description:PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions

| PRJNA657425 | ENA

Chemotranscriptomic Profiling with a Thiamine Monophosphate Photoaffinity Probe

Project description:RNA is a multifaceted biomolecule with numerous biological functions and can interact with small molecule metabolites as exemplified by riboswitches. Here, we profile the Escherichia Coli transcriptome on interactions with the metabolite Thiamine Monophosphate (TMP). We designed and synthesized a photoaffinity probe based on the scaffold of TMP and applied it to chemotranscriptomic profiling. Using next-generation RNA sequencing, several potential interactions between bacterial transcripts and the probe were identified. A remarkable interaction between the TMP probe and the well-characterized ribB riboswitch was validated by RT-qPCR, and further verified with competition assays. Localization of the photocrosslinked nucleotides using reverse transcription and docking predictions of the probe suggested binding to the riboswitch aptamer. After examining binding of unmodified TMP to the riboswitch using SHAPE, we found selective yet moderate binding interactions, potentially mediated by the phosphate group of TMP. Lastly, TMP appeared to enhance gene expression of a reporter gene that is under riboswitch control, while the natural ligand Flavin Mononucleotide (FMN) displayed an inhibitory effect, hinting at a potential biological role of TMP. This work showcases the possibility of chemotranscriptomic profiling to identify new RNA-small molecule interactions.

2025-02-17 | GSE277048 | GEO

Sulfoxide-diazirine (SODA) as a cleavable photoaffinity group to enable identification of photolabeled modification sites via LC-MS3

Project description:Photoaffinity labeling is a common approach in chemical proteomics for identification of small molecule probe tar-gets. However, the modification site on the target protein remains difficult to identify, because of the probe modifica-tion and potential fragmentation during tandem mass spectrometry. We here introduce MS-cleavable photoaffinity groups for a better identification of the modification site. These are based on diazirine photoreactive groups and sul-foxide as MS-labile linker. We have applied these in photoaffinity probes based on the peptide-like aspartic protease inhibitor pepstatin A, and we demonstrate that we can identify and map binding hotspots on structural models of a target protease.

2025-08-25 | PXD065837 | Pride

Ethynylation of Cysteines from Peptides to Proteins in Living Cells

Project description:Efficient methods to introduce bioorthogonal groups, such as terminal alkynes, into biomolecules are important tools for chemical biology. State-of-the-art approaches are based on the introduction of a linker between the targeted amino acid and the alkyne, and still present limitations of either reactivity, selectivity or adduct stability. Herein, we present a new ethynylation method of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. In contrast to other approaches, the acetylene group is directly introduced onto the thiol group of cysteine and can be used in one-pot in a copper-catalyzed alkyne-azide cycloaddition (CuAAC) for further functionalization. Labeling proceeded with reaction rates comparable or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab. Under optimized conditions, high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed a much-improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine-reagent based probes. Fine-tuning of the EBX reagents allowed optimization of their reactivity and physical properties for the desired application.

2020-04-07 | PXD018165 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data