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PLoS One. 2016 Aug 29;11(8):e0161930. doi: 10.1371/journal.pone.0161930. eCollection 2016.

Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

Author information

1
Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, CA, 92010, United States of America.

Abstract

Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells.

PMID:
27571227
PMCID:
PMC5003356
DOI:
10.1371/journal.pone.0161930
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

This study was funded by Ionis Pharmaceuticals, Inc. The funder provided support in the form of salaries for authors TAV and STC but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

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