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

The significance of this protocol is that it provides a means for keeping DNA-encoded sensors functional by immobilizing DNA plasmid templates into semipermeable biocompatible microcapsules. The proposed DNA immobilization technique is versatile. It allows the fabrication of multifunctional biosensors with different in vitro activation mechanisms.

We envision these microcapsules, coupled with the proper sensing elements, could be used to study different biological processes, including the release of signaling molecules in different environments such as biofilms and organs. This technique can provide insights into the biological systems at the microscale, particularly how the immobilization of DNA and molecular crowding can affect the kinetics and mechanism of gene activation. Start with the deposition of the polyethyleneimine prime layer onto the sacrificial silica, or SiO2, microparticles by adding one milliliter of polyethyleneimine solution to 300 microliters of the microparticles in a two-milliliter microcentrifuge tube.

Agitate the mixture on a ThermoMixer at 800 rotations per minute at ambient conditions. Collect the microparticles by centrifugation at 0.2 g for one minute at room temperature, and carefully remove the supernatant. After 15 minutes, wash the microparticles four times with one milliliter of DNase-and RNase-free deionized water by centrifugation at 0.2 g for one minute at room temperature, discarding the supernatant after each centrifugation.

Next, adjust the concentration of DNA plasmids from 50 to 200 nanogram per microliters with DNase-and RNase-free distilled water, and use one milliliter of the prepared solutions to deposit the DNA on the polyethyleneimine-primed microparticles. Gently agitate the microparticle mixture on a ThermoMixer at four degrees Celsius and 800 rpm for 15 minutes, and then collect the microparticles by centrifugation at 0.2 g for one minute. Mark the tubes for DNA plasmids encoding theophylline riboswitch, coupled with GFPa1 as ThyRS-GFPa1, and DNA plasmids encoding Broccoli aptamer as BrocApt.

Carefully remove the supernatant, and wash the microparticles four times with one milliliter of DNase-and RNase-free distilled water as previously demonstrated, discarding the supernatant after each centrifugation. To deposit the silk fibroin layer, add one milliliter of the reconstituted aqueous silk fibroin solution to the DNA-absorbed microparticles, and gently vortex agitate the mixture on the ThermoMixer at 750 rpm for 15 minutes at 10 degrees Celsius. Collect the microparticles by centrifugation, and then wash them once with one milliliter of DNase-and RNase-free distilled water, as explained before.

Repeat the centrifugation, and discard the supernatant. Add 0.5 milliliters of DNase and RNase distilled water, and mix the microparticles by vortexing. Then, treat the microparticles with 0.5 milliliters of 100%methanol at 10 degrees Celsius for five minutes on a ThermoMixer.

After collecting the microparticles by centrifugation, treat the particles with 100%methanol on the ThermoMixer at 750 rotations per minute for 10 minutes at 10 degrees Celsius for beta-sheet formation. Centrifuge the mixture at 0.2 g for one minute at four degrees Celsius to collect microparticles before washing the particles twice with one milliliter of DNase-and RNase-free distilled water as demonstrated before. Dissolve SiO2 cores of microparticles by adding 8%hydrofluoric acid solution to pelleted core shell microparticles.

Transfer the dissolved particles solutions to dialysis devices, and dialyze them against deionized water in a beaker. Then, use a one-milliliter pipette to collect the silk microcapsules suspension from dialysis devices into new two-milliliter microcentrifuge tubes. Transfer 100 microliters of the microcapsule sample into a single well of eight-well chambered glass slides, and allow the capsules to sediment for 20 to 30 minutes prior to imaging using a confocal laser scanning microscope, or CLSM.

Pipette 100 microliters of the suspension of capsules into each well of a chambered glass slide. To each well, sequentially add 300 microliters of the fluorophore solution of varying molecular weights from the lowest to the highest. Mix the mixture by pipetting up and down, and let the mixture incubate for one hour at room temperature until the diffusion of fluorophore solutions reaches equilibrium.

Later, transfer the slide to a CLSM to image each well using a 100x oil immersion objective at an excitation wavelength of 488 nanometers. Identify the area of interest by adjusting the focal plane, and focus on the capsules that appear in the form of circles of the largest diameter. Perform in vitro transcription/translation reaction by sequentially adding the cell-free components to a sample of microcapsules and incubating the tube at 30 degrees Celsius for four hours.

Check the fluorescence on a plate reader using excitation wavelength at 488 nanometers and emission for GFP/FITC filter at around 510 nanometers. Finally, image the silk microcapsules on the CLSM system using lasers at 488 and 561 nanometers. In the study, robust silk fibroin microcaps with a homogeneous size of around 4.5 micrometers and a shell thickness of approximately 500 nanometers were produced.

The permeability of the hollow silk fibroin capsule shells was analyzed by following the molecular weight cutoff method. An increase in the concentration of a polyethyleneimine prime layer leads to increased colloidal stability of microcapsules with more permeable shells, while eliminating the prime layer causes aggregation of capsules with less permeable shell membranes. The GFPa1 expression kinetics during ThyRS activation revealed higher GFPa1 gene activation from DNA plasmids loaded into silk microcapsules than control non-encapsulated DNA.

In the representative analysis, CLSM images of silk fibroin capsules loaded with 32 DNA copies per capsule before and after incubation in the in vitro transcription, translation system are displayed. The cross-sectional intensity profiles of the silk fibroin capsules demonstrated the expression of GFPa1. The transcription kinetics of 20 layered silk microcapsules loaded with 30 DNA copies per capsule was compared with control non-encapsulated DNA.

The higher output signal from silk microcapsules than non-encapsulated DNA samples of equivalent concentrations confirmed the effectivity of silk microcapsules. Following this protocol, several high-resolution microscopy techniques can be applied to image DNA entanglement in the immobilized state and decoupled gene activation process in vitro. This technique can be applied to answer molecular-level dynamics in minimal artificial cells.

Specifically, when coupled with proper gene elements, the microcapsules can be used to study cell-to-cell communication.