Designing DNA With Tunable Regulatory Activity Using Discrete Diffusion.

Designing regulatory DNA with tunable and context-specific activity is a major goal in biotechnology and medicine. Deep generative models offer a promising route for sequence design, yet it remains unclear whether synthetic sequences faithfully recapitulate the motif organization and functional specificity of natural regulatory DNA. Here we present DNA Discrete Diffusion (D3), a generative model that designs regulatory DNA through an iterative nucleotide-substitution process. Across computational benchmarks, D3 improves regulatory sequence generation relative to matched diffusion baselines, producing sequences that more closely match target activity, activity distributions, and sequence composition. In K562 lentiMPRA experiments, D3-designed sequences retained measurable regulatory activity and more closely recapitulated the activity distribution of genomic regulatory sequences than matched diffusion baselines. D3 performs robustly with limited training data and generates sequences informative enough to improve predictive models when labeled data are scarce. When trained without activity labels on task-specific regulatory sequence sets, D3 learns frozen sequence representations that are predictive of enhancer activity and compare favorably to several off-the-shelf genomic language model embeddings. Analysis of the sampling process identifies reproducible phases of sequence exploration, compositional refinement, and motif-associated convergence, providing an interpretable view of how D3 constructs regulatory sequences. These results establish D3 as a practical framework for designing synthetic regulatory DNA and studying sequence features associated with context-specific regulatory activity.