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

SUBMITTER: Sau SP 

PROVIDER: S-EPMC3505983 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature