A FRET-based screening assay for nucleic acid ligands

Area of Science:

• Molecular Biology
• Biochemistry
• Drug Discovery

Background:

• Specific DNA/RNA secondary structures in genes can alter protein expression, contributing to serious diseases.
• These structures can form in promoter regions, coding sequences, or other genomic areas like telomeres.
• Compounds interacting with these structures offer potential for disease diagnosis and treatment.

Purpose of the Study:

• To present a novel FRET melting assay for evaluating compound libraries.
• To assess the ability of compounds to stabilize nucleic acid secondary structures.

Main Methods:

• A FRET melting assay was developed using model oligonucleotides labeled with fluorescent donor (FAM) and acceptor (TAMRA).
• The assay measures changes in FAM fluorescence intensity with temperature to determine melting temperatures (Tm).
• Compounds are tested for their ability to increase Tm, quantified as ΔTm, indicating stabilization of nucleic acid structures.

Main Results:

• The FRET melting assay successfully determined Tm for four different nucleic acid structures.
• The assay quantifies compound-induced stabilization via the ΔTm parameter.
• The method is adaptable to high-throughput screening formats (96-well and 384-well plates).

Conclusions:

• The developed FRET melting assay is an effective tool for screening compounds that stabilize nucleic acid structures.
• This assay can accelerate the discovery of novel diagnostics and therapeutics targeting structure-related diseases.
• The assay's adaptability facilitates large-scale compound library screening.