Experimental and Computational Dataset for ""Direct monitoring of the thermodynamics and kinetics of DNA and RNA dinucleotide hybridization onto gaps and overhangs"

Binding of short nucleic acid segments (<4 nucleotides) to single-strand templates occurs as a critical intermediate in processes such as non-enzymatic nucleic acid replication and toehold-mediated strand displacement. The templates in these reactions often contain adjacent duplex segments that stabilize base pair formation to single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood due to the
experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide
hybridization using steady-state and temperature-jump infrared spectroscopy. Thermodynamic results indicate that duplex segments adjacent to overhangs and gaps stabilize dinucleotide binding
through a combination of coaxial stacking interactions and additional factors that remain unclear. All-atom molecular dynamics simulations indicate potential structural differences between the
dinucleotide-gap complexes and canonical duplexes that may contribute to binding stability. We demonstrate time-resolved measurements of dinucleotide dehybridization and observe timescales ranging from 200 ns to 40 µs depending on the template and temperature. Together, our work provides an initial step for predicting the binding stability and kinetics of short DNA and RNA duplex segments onto various type of nucleic acid templates