Impact of single mutations on binding kinetics oftriplex forming oligos revealed by fluorescence proximitysensing in heliX(R) biosensor

Abstract

The sequence specific association of RNA with DNA via formation of RNA/DNA triple helices is relevant for regulation of gene expression, repair mechanisms, and chromatin organization. Formation of such RNA/DNA triplexes requires poly-purine sequences, which allow for Hoogsteen base-paring in addition to Watson-Crick pairing in the major groove of DNA. Binding of triplex forming oligos (TFOs) is sequence-specific and understanding sequence dependencies is key for the development of targeted tools for gene therapy. Here, we highlight a direct approach for determining binding kinetics and binding constants for TFOs using the state-of-the-art heliX(R)biosensor tool. With this, we provide key insights into the binding kinetics of RNA and DNA TFOs to a triplex targeting site (TTS)-containing DNA double helix measured in real-time. Dependent on the introduced base, point mutations in one position of a triplex forming oligo (TFO) can change the dissociation constant (KD) by several orders of magnitude or just by one log, affecting primarily the dissociation rate. Furthermore, we demonstrate that the heliX(R)biosensor assay is also well-suited for detection of rather weak triplex formation. The weakest binding we could identify was 140 M, for a TFO, which other studies considered as non-binding.