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

Area of Science:

• Biotechnology
• Materials Science
• Synthetic Biology

Background:

• Liposomal vesicles are common for encapsulating transcription-translation systems but have limitations in permeability and functionalization.
• Artificial cells offer potential for biomolecule synthesis, biosensing, and delivery.
• Layer-by-layer (LbL) assembly with natural polymers presents an alternative to liposomes for microcapsule fabrication.

Purpose of the Study:

• To design and fabricate DNA-laden silk fibroin microcapsules for artificial cell applications.
• To investigate the effect of LbL assembly parameters on microcapsule properties and DNA immobilization.
• To demonstrate the utility of these microcapsules for biosensing and biomolecule synthesis.

Main Methods:

• Fabrication of microcapsules using layer-by-layer (LbL) assembly of regenerated silk fibroin.
• Immobilization of DNA templates encoding riboswitches and RNA aptamers within the microcapsule membrane.
• Optimization of LbL parameters including polymer primer, silk concentration, and DNA loading.
• Functionalization of microcapsule shells with gold nanoparticles (AuNPs) and antibodies (IgG).
• Assessment of microcapsule permeability, DNA bioactivity, and protein synthesis via in vitro transcription/translation.

Main Results:

• Biocompatible, semipermeable, and DNA-laden silk microcapsules were successfully prepared.
• Immobilized DNA within the capsule membrane enhanced stability and sensing element output.
• Selective permeability of silk microcapsules facilitated cell-free system component diffusion and biomolecule synthesis (mRNA, GFPa1 protein).
• Functionalization with AuNPs and IgG demonstrated potential for remote sensing and targeted delivery.

Conclusions:

• Silk fibroin microcapsules provide a promising platform for creating artificial cells with enhanced stability and functionality.
• The selective permeability and immobilization capabilities are crucial for efficient biosensing and biomolecule production.
• These multifunctional microcapsules hold potential for advancing multiplexed biosensors and targeted delivery systems.