title: A FRET-based screening assay for nucleic acid ligands.
abstract: Some of the most serious diseases are characterized by the presence of a specific secondary structure within DNA or RNA, often in the promoter or the coding region of the responsible gene, that enhances or disrupts expression of the protein. Structural elements that impact cellular function may also be formed in other genomic regions such as telomeres. Compounds that interact with such structural elements may be useful in diagnosis or treatment of patients. In this report, we present a FRET melting assay that allows testing of libraries of compounds against four different nucleic acid structures. Compounds are tested to determine whether they stabilize preformed secondary structures (i.e., whether they cause an increase in melting temperature (T(m))). This property is described by the ΔT(m) parameter, which is the difference between the T(m) of the compound-stabilized structure and the T(m) of the unbound structure. Model oligonucleotides are labeled with FAM as a fluorescent donor and TAMRA as an acceptor. The intensity of FAM fluorescence is recorded as a function of temperature. Melting temperatures are determined by the FRET method in 96-well plates; this assay could easily be converted into 384-well format.
authors.affiliation: INSERM, U869, ARNA Laboratory, European Institute of Chemistry and Biology, 2 rue Robert Escarpit, F-33600 Pessac, France. renciuk@ibp.cz
authors.forename: Daniel
authors.initials: D
authors.lastname: Renčiuk
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.forename: Lionel
authors.initials: L
authors.lastname: Beaurepaire
authors.forename: Aurore
authors.initials: A
authors.lastname: Guédin
authors.forename: Anne
authors.initials: A
authors.lastname: Bourdoncle
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Mergny
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D054856:G-Quadruplexes; D006801:Humans; D008024:Ligands; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D054852:Small Molecule Libraries; D016615:Telomere / Q000737:chemistry*
chemical_list: D008024:Ligands; D009696:Nucleic Acids; D009841:Oligonucleotides; D054852:Small Molecule Libraries