Protein and DNA co-crystals are most commonly prepared to reveal structural and functional details of DNA-binding proteins when subjected to X-ray diffraction. However, biomolecular crystals are notoriously unstable in solution conditions other than their native growth solution. To achieve greater application utility beyond structural biology, biomolecular crystals should be made robust against harsh conditions. To overcome this challenge, we optimized chemical DNA ligation within a co-crystal. Co-crystals from two distinct DNA-binding proteins underwent DNA ligation with the carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) under various optimization conditions: 5′ vs. 3′ terminal phosphate, EDC concentration, EDC incubation time, and repeated EDC dose. This crosslinking and DNA ligation route did not destroy crystal diffraction. In fact, the ligation of DNA across the DNA–DNA junctions was clearly revealed via X-ray diffraction structure determination. Furthermore, crystal macrostructure was fortified. Neither the loss of counterions in pure water, nor incubation in blood serum, nor incubation at low pH (2.0 or 4.5) led to apparent crystal degradation. These findings motivate the use of crosslinked biomolecular co-crystals for purposes beyond structural biology, including biomedical applications.
High-precision nanomaterials to entrap DNAbinding molecules are sought after for applications such as controlled drug delivery and scaffold-assisted structural biology. Here, we engineered protein−DNA cocrystals to serve as scaffolds for DNA-binding molecules. The designed cocrystals, isoreticular cocrystals, contain DNA-binding protein and cognate DNA blocks where the DNA−DNA junctions stack end-to-end. Furthermore, the crystal symmetry allows topology preserving (isoreticular) expansion of the DNA stack without breaking protein−protein contacts, hence providing larger solvent channels for guest diffusion. Experimentally, the resulting designed isoreticular cocrystal adopted an interpenetrating I222 lattice, a phenomenon previously observed in metal−organic frameworks (MOFs). The interpenetrating lattice crystallized dependably in the same space group despite myriad modifications at the DNA−DNA junctions. Assembly was modular with respect to the DNA inserted for expansion, providing an interchangeable DNA sequence for guest-specified scaffolding. Also, the DNA−DNA junctions were tunable, accommodating varied sticky base overhang lengths and terminal phosphorylation. As a proof of concept, we used the interpenetrating scaffold crystals to separately entrap three distinct guest molecules during crystallization. Isoreticular cocrystal design offers a route to a programmable scaffold for DNA-binding molecules, and the design principles may be applied to existing cocrystals to develop scaffolding materials.
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