preparation-multifunctional-silk-based-microcapsules-loaded-with-dna

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

UES Inc.

Wright-Patterson AFB

Assembling synthetic bioparts into simplified artificial cells holds tremendous promise for advancing studies into the synthesis, biosensing, and delivery of biomolecules. Currently, the most successful techniques for encapsulation of the transcription-translation machinery exploit compartmentalization in liposomal vesicles. However, improvements to these methods may increase permeability to polar molecules, functionalization of the membrane with biologically active elements, and encapsulation efficiency. Microcapsules prepared  templated layer-by-layer (LbL) assembly using natural polymers have the potential to resolve some of the hurdles associated with liposomes. Here, we introduce a design for immobilizing DNA templates encoding translationally activated riboswitches and RNA aptamers into microcapsules prepared from regenerated silk fibroin protein. Adjusting several key parameters such as the presence of a polymer primer, concentration of silk protein, and DNA loadings during LbL assembly resulted in biocompatible, semipermeable, DNA-laden microcapsules. To preserve bioactivity, DNA was immobilized inside of the capsule membrane, which not only promoted stability during long-term storage at ambient conditions but also improved output response from spatially confined DNA-encoded sensing elements (SEs). Multiple copies of mRNA and GFPa1 protein were synthesized upon activation with specific analytes during  transcription/translation reactions, demonstrating that selective permeability of silk microcapsules was essential for the diffusion of components of the cell-free system inside of the capsules. Further functionalization of capsule shells with gold nanoparticles (AuNPs) and antibodies (IgG) demonstrated the applicability of microcompartmentalized colloidal objects carrying SEs for remote sensing and/or targeted delivery. In the future, multifunctional, biocompatible silk-based microcapsules loaded with different RNA sensors can help advance the design of multiplexed biosensors tracking multiple biomarkers in complex media.

Improving the Activity of DNA-Encoded Sensing Elements through Confinement in Silk Microcapsules.

Strategies for the encapsulation of cells for the design of cell-based sensors require efficient immobilization procedures while preserving biological activity of the reporter cells. Here, we introduce an immobilization technique that relies upon the symbiotic relationship between two bacterial strains: cellulose-producing  cells; and recombinant  cells harboring recombinase-based dual-color synthetic riboswitch (RS), as a model for cell-based sensor. Following sequential coculturing of recombinant cells in the cellulose matrix, final immobilization of  cells was completed after reconstituted silk fibroin (SF) protein was added to a "living membrane" generating the composite bacterial cellulose-silk fibroin (BC-SF) scaffold. By controlling incubation parameters for both types of cells, as well as the conformations in SF secondary structure, a variety of robust composite scaffolds were prepared ranging from opaque to transparent. The properties of the scaffolds were compared in terms of porosity, water capacity, distribution of recombinant cells within the scaffolds matrix, onset of cells activation, and ability to protect recombinant function of cells against UV irradiation. The closer-fitted microstructure of transparent BC-SF scaffolds resulted in leakage-free encapsulation of recombinant cells with preserved RS function because of a combination of several parameters that closely matched properties of a biofilm environment. Along with proper elasticity, fine porosity, capacity to retain the water, and ability of SF to absorb UV light, the composite hydrogel material provided necessary conditions to form confined cell colonies that modified cell metabolism and enhanced cell resilience to the stresses induced by encapsulation.

Irina Drachuk

UES Inc.

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches