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A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites

ABSTRACT: A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites

PROVIDER: PRJNA707022 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13865128_1.fastq.gz	Fastqsanger.gz
	SRR13865128_2.fastq.gz	Fastqsanger.gz
	SRR13865129_1.fastq.gz	Fastqsanger.gz
	SRR13865129_2.fastq.gz	Fastqsanger.gz
	SRR13865130_1.fastq.gz	Fastqsanger.gz

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Similar Datasets

A Chemical Probe Based on the PreQ1 Metabolite Enables Transcriptome-wide Mapping of Binding Sites

Project description:We report the optimization and application of diazirine photocrosslinking probes that enable selective covalent crosslinking to the PreQ1 RNA aptamer and any other interacting RNAs in close proximity. By evaluating the impact of the linker structure and length on crosslinking efficiency we were able to shed important light on what factors have the largest impact on photocrosslinking efficiency in this model system. Additionally, the best compound (compound 11) was shown to efficiently and selectively crosslink site-specifically to the PreQ1 aptamer even in the presence of competing human cellular RNAs. Sequencing experiments were performed in human MCF-7 cell total RNA both with and without spike in of exogenous PreQ1 aptamer. In the aptamer spike-in samples, the only RNA target that was significantly enriched after differential expression analysis with the negative control (compound 17) was the PreQ1 aptamer suggesting that this compound does in fact selectively crosslink to the desired target even in the presence of other RNAs. Additionally, in the samples without spike-in of the aptamer we were able to identify 16 significantly enriched hit RNAs that may represent novel metabolite-RNA interactions. Competitive RT-qPCR experiments confirmed enrichment of a handful of select genes with 11 and showed that the free PreQ1 ligand, which lacks the diazrine crosslinking moeity, is able to compete away compound 11. This suggests that these interactions are specific.

2021-08-09 | GSE168353 | GEO

A chemical probe based on the PreQ<sub>1</sub> metabolite enables transcriptome-wide mapping of binding sites.

Project description:The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.

| S-EPMC8494917 | biostudies-literature

Dual Chemical Labeling Enables Nucleotide-Resolution Mapping of DNA Abasic Sites and Common Alkylation Damage in Human Mitochondrial DNA

Project description:Understanding the genomic locations of DNA modifications is pivotal for deciphering their roles in gene regulation, mutagenesis, and pathology. The affinity probe-based enrichment of lesion-containing DNA represents a key strategy for sequencing DNA modifications. Existing methods are limited in the specificity of labeling and enrichment of abasic (AP) sites, a prevalent DNA modification and repair intermediate. Herein, we devise a novel approach, termed dual chemical labeling-assisted sequencing (DCL-seq), for mapping AP sites. DCL-seq features two designer compounds for enriching and mapping AP sites specifically at single-nucleotide resolution. For proof of principle, we mapped AP sites in mitochondrial DNA (mtDNA) from cultured human cells under different biological conditions. Also, we demonstrated the broader applicability of the method in sequencing additional DNA modifications in mtDNA, such as N7-methyl-2'-deoxyguanosine and N3-methyl-2'-deoxyadenosine, when coupled with a lesion-specific repair enzyme. Together, DCL-seq holds the promise to sequence multiple DNA modifications in various biological samples.

2022-09-24 | GSE213585 | GEO

Function-first proteomic strategies for chemical probe discovery in human cells

Project description:Function-first proteomic strategies for chemical probe discovery in human cells

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Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

Project description:Probe-Seq is a method that allows transcriptional profiling of specific cell types from heterogeneous tissue by labeling RNA. Briefly, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated based on RNA abundance of defined markers. We applied Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas as well as the Drosophila gut. We also showed that Probe-Seq is compatible with frozen nuclei and that multiplexed Probe-Seq enables iterative isolation of multiple cell types from the same tissue sample. Provided that unique markers are available, this simple, novel method could potentially enable bulk RNA sequencing of any cell type from any organism.

2019-12-09 | GSE135572 | GEO

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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.

2021-08-06 | PXD017641 | Pride

A chemical probe to monitor parkinsonism-associated protein DJ-1 in live cells

Project description:Oxidative stress is a contingent trigger of Parkinson`s disease (PD). DJ-1, a protein involved in oxidative stress sensing, gained major attention when mutations in its gene were identified in PD patients. Although the study of the corresponding protein function is still in its infancy, a crucial oxidation sensor at a conserved cysteine was linked to the disease. Inspired by inhibition studies with a bacterial homolog of DJ-1 we here screen several amino-epoxycylcohexenones and identify a chemical probe that exhibits specificity for the human protein in various cell lines. The probe selectively labeled the oxidation sensor via nucleophilic attack of the conserved cysteine onto the epoxide ring. This covalent addition occurred only in the reduced state. Whole proteome studies in HeLa, A549 and SHSY5Y cell lines confirmed strong enrichment of solely reduced DJ-1 which was diminished by increasing oxidative stress. The probe thus facilitates the first selective in situ monitoring of DJ-1 in its reduced state and enables studying the function of this important biomarker in dependence of oxidative stress.

2018-07-17 | PXD009444 | Pride

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Project description:Cellular selectivity of a chemical probe for the UBD of HDAC6. A biotinylated analog of HDAC6 chemical probe SGC-UBD253 bound to streptavidin beads was used to enrich HDAC6 from cytoplasmic fraction of HEK293 lysate. A triplicate of each condition - competition with probe, control, DMSO - was done.

2023-10-24 | PXD039880 | Pride

Dual Chemical Labeling Enables Nucleotide-Resolution Mapping of DNA Abasic Sites and Common Alkylation Damage in Human Mitochondrial DNA

Project description:Dual Chemical Labeling Enables Nucleotide-Resolution Mapping of DNA Abasic Sites and Common Alkylation Damage in Human Mitochondrial DNA

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