Intrinsically disordered protein droplet-enhanced oligonucleotide assembly enables rapid oligonucleotide-to-protein expression.

Recent advances in in silico protein design and bioinformatics have enabled the rapid generation of candidate sequences for functional proteins. However, experimental validation remains a bottleneck, largely due to time-consuming DNA assembly and cell-based cloning processes. Technologies that reduce the time required to convert synthetic oligonucleotides (oligos) into expressed proteins are therefore of considerable interest. Here, we demonstrate that phase-separated droplets formed by the intrinsically disordered protein (Ddx4N1) concentrate both oligos and ligation enzymes, enabling efficient oligo assembly at nanomolar to sub-nanomolar concentrations that are typically inaccessible to conventional ligation-based methods. The assembled products can be directly introduced into femtoliter-scale microreactors for digital cell-free gene expression, allowing protein expression from single assembled DNA molecules without polymerase chain reaction amplification or cellular cloning. Simultaneous expression of two distinct proteins from separately assembled DNA templates in a one-pot reaction was also demonstrated. The complete workflow-from oligo assembly to detectable protein expression-can be performed within half a day. While further development will be required to enhance reaction parallelization and enable systematic retrieval of sequence information from expressed products, this amplification-free, low-input system establishes a technical foundation for integrating oligo-pool-based gene assembly with digital protein prototyping platforms.