Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA260045 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1562560_1.fastq.gz | Fastqsanger.gz | |||
| SRR1562560_2.fastq.gz | Fastqsanger.gz | |||
| SRR1562561_1.fastq.gz | Fastqsanger.gz | |||
| SRR1562561_2.fastq.gz | Fastqsanger.gz | |||
| SRR1562562_1.fastq.gz | Fastqsanger.gz |
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Project description:Targeting cellular RNA by small molecules has come to the forefront of biotechnology and holds great promise for therapeutic use. Strategies to identify, validate and optimize these molecules are essential, but are still lacking in some aspects. In particular, the site-specific covalent labeling and modification of RNA in living cells poses many challenges. Here, we describe a general structure-guided approach to engineer non-covalent RNA aptamer–ligand complexes into their covalent counterparts using a molecular tether. The key is to modify the native ligand with an electrophilic handle that allows it to react specifically with a guanine at the RNA ligand binding site. We show that site-specific cross-linking between ligand and RNA is achieved in mammalian cells upon transfection of a genetically encoded version of the preQ1-I riboswitch aptamer. Further, we showcase the versatility of the tether by engineering the first covalent fluorescent light-up aptamer (coFLAP) out of the non-covalent Pepper FLAP. The coPepper system maintains strong fluorescence in live-cell imaging even after repeated washing. Thus, any background signal arising from unspecific fluorophore accumulation in the cell can be eliminated. In addition, we generated a bifunctional Pepper ligand containing a second handle for bioorthogonal chemistry to allow for easily traceable and efficient pulldown of the covalently linked target RNA. Finally, we provide evidence for the suitability of this tethering strategy for specific drug targeting. Taken together, our results show that functionalized ligands generated by rational design can cross-link site-specifically with target RNAs in cells, and hence, open up a wide range of applications in RNA biology that require irreversible small molecule binding.
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