Nanometer-sized features and molecular recognition properties make DNA a useful material for nanoscale construction, but degradation in biological fluids poses a considerable roadblock to biomedical applications of DNA nanotechnology. Here, we report the remarkable biostability of a multistranded motif called paranemic crossover (PX) DNA. Compared to double stranded DNA, PX DNA has dramatically enhanced (sometimes >1000 fold) resistance to degradation by four different nucleases, bovine and human serum, and human urine. We trace the cause of PX's biostability to DNA crossovers, showing a continuum of protection that scales with the number of crossovers. These results suggest that enhanced biostability can be engineered into DNA nanostructures by adopting PX-based architectures or by strategic crossover placement.

The detection of molecular biomarkers plays a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. The ability to detect multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. One promising approach is the use of dynamic DNA nanostructures

Base stacking interactions between adjacent bases in DNA and RNA are important for many biological processes and in biotechnology applications. Previous work has estimated stacking energies between pairs of bases, but contributions of individual bases has remained unknown. Here, we use a Centrifuge Force Microscope for high-throughput single molecule experiments to measure stacking energies between adjacent bases. We found stacking energies strongest between purines (G|A at −2.3 ± 0.2 kcal/mol) and weakest between pyrimidines (C|T at −0.5 ± 0.1 kcal/mol). Hybrid stacking with phosphorylated, methylated, and RNA nucleotides had no measurable effect, but a fluorophore modification reduced stacking energy. We experimentally show that base stacking can influence stability of a DNA nanostructure, modulate kinetics of enzymatic ligation, and assess accuracy of force fields in molecular dynamics simulations. Our results provide insights into fundamental DNA interactions that are critical in biology and can inform design in biotechnology applications.