Enzyme- and DNAzyme-Driven Transient Assembly of DNA-Based Phase-Separated Coacervate Microdroplets

An assembly of dissipative, transient, DNA-based microdroplet (MD) coacervates in the presence of auxiliary enzymes (endonucleases and nickases) or MD-embedded DNAzyme is introduced. Two pairs of different Y-shaped DNA core frameworks modified with toehold tethers are cross-linked by complementary toehold-functionalized duplexes, engineered to be cleaved by EcoRI or HindIII endonucleases, or cross-linked by palindromic strands that include pre-engineered Nt.BbvCI or Nb.BtsI nicking sites, demonstrating transient evolution/depletion of phase-separated MD coacervates. By mixing the pairs of endonuclease- or nickase-responsive MDs, programmed or gated transient formation/depletion of MD frameworks is presented. In addition, by cross-linking a pre-engineered Y-shaped core framework with a sequence-designed fuel strand, phase separation of MD coacervates with embedded Mg²⁺-DNAzyme units is introduced. The DNAzyme-catalyzed cleavage of a ribonucleobase-modified hairpin substrate, generating the waste product of the metabolite fragments, leads to the metabolite-driven separation of the cross-linked coacervates, resulting in the temporal evolution and depletion of the DNAzyme-functionalized MDs. By employing a light-responsive caged hairpin structure, the light-modulated fueled evolution and depletion of the DNAzyme-active MDs are presented. The enzyme- or DNAzyme-catalyzed transient evolution/depletion of the MD coacervates provides protocell frameworks mimicking dynamic transient processes of native cells. The possible application of MDs as functional carriers for the temporal, dose-controlled release of loads is addressed.