Researchers at UCLA have developed a two-color fluorescent reporter assay for high-throughput detection of compounds that affect alternative pre-mRNA splicing in individual living cells, heterogeneous cell populations and live animals.
Prior to translation, transcription generates a precursor molecule (pre-mRNA) that contains both introns (intervening sequences) and exons (protein coding regions). Alternative splicing pathways vary the production of a mature mRNA strand by modifying the introns removed and the exons joined. Depending on the splice sites, these mRNA variances give rise to proteome diversity by changing the encoded protein structure, which in turn can affect ligand binding, allosteric regulation, protein localization, etc. Although mutations in splice signals account for 15% of genetic diseases caused by point mutations indicating a pressing need for research into the mechanisms controlling alternative splicing, experimental efforts to discover compounds targeting splicing are hampered by a lack of reliable, reproducible, and high-throughput techniques.
Researchers at UCLA have developed a reporter assay for use in the detection of compounds that modulate alternative splicing. This novel and robust two-color fluorescent system provides a means to distinguish between general changes in splicing, such as efficiency, accuracy and transcription/translation abnormalities, and a particular alternative splicing event.
The invention has been tested as a high-throughput application (96- and 384-well plates) with a variety of fully functioning prototypes. Preliminary experiments indicate that the assay scales well to 1536 well format. Its use at UCLA has already revealed several compounds that modulate alternative splicing and can lead to development of therapy for Spinal Muscular Atrophy.
|United States Of America||Issued Patent||8,580,511||11/12/2013||2006-704|
biomedical, drug discovery, drug screening, gene therapy, genomics/genetics, proteomics, alternative splicing, pre-mRNA, mRNA