title: Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation--a case study using triplex-forming LNAs.
abstract: Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (K(eq)) and association/dissociation rate constants (k(on) and k(off)). The 'fluorescent intercalator displacement replacement' (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson-Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine.
authors.affiliation: Department of Chemistry, University of Idaho, PO Box 442343, Moscow, ID 83844-2343, USA.
authors.forename: Sujay P
authors.initials: SP
authors.lastname: Sau
authors.forename: Pawan
authors.initials: P
authors.lastname: Kumar
authors.forename: Pawan K
authors.initials: PK
authors.lastname: Sharma
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Hrdlicka
mesh_terms: D004247:DNA / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D007364:Intercalating Agents / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D005456:Fluorescent Dyes; D007364:Intercalating Agents; D009841:Oligonucleotides; C477371:locked nucleic acid; C051802:triplex DNA; D004247:DNA; D004996:Ethidium