Synthetic DNA motifs form the basis of nucleic acid nanotechnology, and their biochemical and biophysical properties determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.