A modular DNA toolkit for single-molecule biophysics

Long, customizable DNA molecules are an essential tool for in vitro single-molecule biophysics. Most studies still rely on the {lambda}-phage genome, which has a fixed sequence, a non-uniform GC distribution, and limited options for modification. To overcome these limitations, we developed a modular DNA (ModDNA) toolkit that integrates in silico sequence design with cloning, to create long (up to 100 kb) DNA molecules with entirely user-defined sequences. We used a computational workflow with curated databases to design a motif-depleted sequence backbone, into which any desired sequence can be inserted at any position. Golden Gate assembly then builds these sequences into constructs, which we extend iteratively in defined increments up to 100 kb. We show precise placement of local features, e.g. a fluorophore, a bound protein, and a DNA-origami structure, and illustrate the construct's utility in single-molecule visualization assays, including DNA loop extrusion by human condensin I. ModDNA offers a scalable, sequence-programmable alternative to {lambda}-DNA and other restrictive substrates. More broadly, the motif-aware feature-free design connects single-molecule biophysics to emerging efforts in machine-guided regulatory-sequence design and synthetic-genome engineering, where controlled programmable sequence context is increasingly essential.