OrthoSeq: A Design Workflow for Thermodynamically Orthogonal DNA Sequence-Pair Libraries

Programmable DNA hybridization underlies many technologies in DNA nanotechnology, fluorescence imaging, and synthetic DNA sequence assembly. A common design challenge is to generate large sequence libraries in which each strand binds strongly to its intended partner while avoiding cross-hybridization and self-folding. Here, we introduce OrthoSeq, a workflow for designing thermodynamically orthogonal DNA sequence-pair libraries under user-defined experimental conditions. OrthoSeq uses NUPACK to evaluate intended binding, cross-hybridization, and self-folding. Within OrthoSeq, candidate sequence pairs form vertices in a conflict graph, while pairwise cross-hybridization conflicts define the edges. Library selection is then formulated as an independent-set problem and addressed using search strategies tailored to the computational regime considered here, in which thermodynamic evaluations dominate the computational cost. In benchmark comparisons, these strategies identify larger sequence-pair libraries than the commonly employed sequential candidate-addition strategy under the same thermodynamic constraints and computational budget. We further show that sequence-level barcode libraries can serve as candidate pools for thermodynamic refinement with OrthoSeq. To support practical use, OrthoSeq provides a graphical user interface that implements the complete workflow. Altogether, OrthoSeq provides an application-agnostic framework for designing DNA sequence-pair libraries under explicit thermodynamic constraints.