Fundamentals on the Kinetic and Thermodynamic Analysis of Oligonucleotide DNA Hybridization by Surface Plasmon Resonance: A Guide for HIF1α Antisense Design.

Abstract

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.