Structural changes of DNA scaffold influence hybridization kinetics of localized DNA four-way junctions

Madysn Roth¹, Andrea Bardales¹, Katherine Taylor¹, Dmitry Kolpashchikov^1,2,3

1. Chemistry Department, University of Central Florida, Orlando, Fl 32816, USA
2. National Center for Forensic Science University of Central Florida, Orlando, Florida 32816, USA
3. Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida
32816, USA

DNA nanostructures are the result of predesigned DNA oligonucleotide assemblies; these nanostructures have been proposed as scaffolds to host chemical reactions, bio-sensing, and drug delivery by using single-stranded DNA (ssDNA) anchored on their surface. Thus, studying the kinetics of localized ssDNA is relevant in understanding the effects of using DNA scaffolds to speed processes dependent on DNA hybridization. ssDNA hybridization into four-way junctions (4WJ) has been proposed in nucleic acid sensors and molecular computing. Therefore, we studied the effects of different structural changes made to a DNA scaffold in the hybridization kinetics of localized 4WJ. Our DNA scaffold is composed of four oligonucleotides: two rails and two staples; its center anchors 4WJ-forming ssDNA. Hybridization kinetics were measured through the binding of a DNA molecular reporter system composed of one ssDNA labeled with a fluorophore and a second ssDNA labeled with a quencher. The reporter system is attached to the DNA scaffold to suppress the influence of diffusion, which is the limiting step. Studies were conducted by adding various double-stranded DNA (dsDNA) bridges within the DNA scaffold. The dsDNA bridges inflict rigidity on the DNA scaffold, thus we modulated the flexibility of its structure. Our results showed that dsDNA bridges influence the initial fluorescence, kinetics, and equilibrium of the hybridizing 4WJ. Additionally, we observed that these effects varied depending on the number and position of dsDNA bridges in the DNA scaffold.