Abstract

The design of synthetic antisense oligonucleotides (ASOs) is improved by considering their thermodynamics. ASOs can be chemically modified with Locked Nucleic Acid (LNA) sugars to increase ASO/RNA duplex stability and phosphorothioate (PS) backbones to increase binding affinity to proteins and resistance towards nuclease degradation. We here present work on fluorescence-based melting-curve analysis of 948 LNA/DNA PS-ASOs to derive enthalpic and entropic changes upon ASO/RNA duplex formation and estimation of nearest-neighbor (NN) parameters. These NN parameters allow ASO/RNA duplex stability to be accurately predicted for any sequence before synthesis and experimental testing which helps identify potent and well-tolerated designs.