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 ...

Wright-Patterson AFB

Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered. A plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. Screening was performed by placing the riboswitch library upstream of the Tobacco Etch Virus (TEV) protease coding sequence in one plasmid; a second plasmid encoded a FRET-based construct linked with a peptide containing the TEV protease cleavage site. Addition of DNT to bacterial culture activated the riboswitch, initiating translation of TEV protease. In turn, the protease cleaved the linker in the FRET-based fusion protein, causing a change in fluorescence. This new riboswitch exhibited a 10-fold increase in fluorescence in the presence of 0.5 mM DNT compared to the system without target.

Development of a 2,4-dinitrotoluene-responsive synthetic riboswitch in E. coli cells.

Riboswitches are RNA-based "sensors" that utilize chemically induced structural changes in the 5'-untranslated region of mRNA to regulate expression of downstream genes. Coupling a specific riboswitch with a reporter gene system translates chemical detection by the cell into a quantifiable reporter protein signal. For the majority of reporter gene systems, the readout signal is only expressed in the presence of the target analyte. This makes it difficult to determine the viability and localization of the uninduced biosensor when it is used for "real-word" applications. To address this problem, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). Thus, the sensor is always fluorescent, but its color is determined by detection of a specific analyte. We demonstrate that the recombinase-based dual-color reporter can be successfully applied to monitor the activation of a theophylline synthetic riboswitch that was used as our model system. To show the feasibility of the FimE recombinase-based system to serve as a reporter for monitoring activation of multiple synthetic riboswitches and, therefore, expand the applicability of the system, we tested a number of previously developed synthetic riboswitches responsive to different analytes. We show that the dual-color reporter system can be successfully used to monitor activation of M6 and M6″ riboswitches responsive to ammeline and pyrimido[4,5-d]pyrimidine-2,4-diamine, respectively, and a 2,4,6-trinitrotoluene-responsive riboswitch developed in this study. We also demonstrate that the system can be reversed by HbiF recombinase-mediated fimS inversion to the initial state of the fluorescent reporter, creating a resettable and reusable cell-based sensor.

Riboswitch-Based Reversible Dual Color Sensor.

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.

Screening and selection of artificial riboswitches.

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.

Svetlana Harbaugh

711th Human Performance Wing,
Airman Systems Directorate,
Air Force Research Laboratory,
711th Human Performance Wing, Airman Systems Directorate, Air Force Research Laboratory

Wright-Patterson AFB

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

Svetlana Harbaugh

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}711th Human Performance Wing,Airman Systems Directorate,Air Force Research Laboratory,711th Human Performance Wing, Airman Systems Directorate, Air Force Research Laboratory

Wright-Patterson AFB

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

711th Human Performance Wing,
Airman Systems Directorate,
Air Force Research Laboratory,
711th Human Performance Wing, Airman Systems Directorate, Air Force Research Laboratory