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.
Highlights
Proteins interact with DNA and RNA through electrostatic interactions, hydrogen bonding, hydrophobic interactions, and base stacking [1,2,3,4]
It was recently reported that the Drosophila behavior/human splicing (DBHS) family proteins P54nrb (NONO), PSF (SFPQ), and PSPC1 can bind to phosphorothioate modified antisense oligonucleotides (PS ASOs), resulting in inhibition of ASO directed RNAseH1-mediated activity [14]
We developed a bioluminescence resonance energy transfer (BRET) affinity assay in which NLuc was fused in frame to P54nrb at either the amino- (NLuc-P54nrb) or carboxy-terminus (P54nrb-NLuc) of the protein under the control of the CMV immediate early enhancer/promoter as detailed in Materials and Methods (Fig 1A)
Summary
Proteins interact with DNA and RNA through electrostatic interactions, hydrogen bonding, hydrophobic interactions, and base stacking [1,2,3,4]. These forces contribute in varying degrees to proteins binding in a structure and sequence specific or non-sequence specific manner [5]. The broad and increasing use of oligonucleotides of various types as research tools and platforms for drug discovery and development demand a more thorough understanding of how these pharmacological agents interact with various proteins and how chemical modifications, sequence and structure influence interactions with proteins. Antibodies must frequently be generated to facilitate isolation
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