This work was supported by LRIR 16RH3003J grant from the Air Force Office of Scientific Research, as well as the Synthetic Biology for Military Environments Applied Research for the Advancement of S&#38;T Priorities (ARAP) program of the U.S. Office of the Under Secretary of Defense for Research and Engineering.
The plasmid vector sequence for ThyRS (pSALv-RS-GFPa1, 3.4 kb) was generously provided by Dr. J. Gallivan. Silkworm cocoons from Bombyx mori were generously donated by Dr. D.L. Kaplan from Tufts University, MA.

1. Construction of plasmid vector.
<ol>
	<li>Construct a plasmid vector (pSALv-RS-GFPa1, 3.4 kb) by amplification of the coding sequence of a theophylline riboswitch (ThyRS) coupled with GFPa1 from pJ201:23976-RS-GFPa1 vector (designed and created by DNA2.0) and insertion into E. coli expression vector, pSAL13. Use forward (5&#39;-CGTGGTACCGGTGATACCAGCATCGTCTTGATG-3&#39;) and reverse (5&#39;-CGTGCTCAGCTTAAGCCAGCTCGTAG-3&#39;) primers to amplify the coding sequence of ThyRS coupled with GFPa1 and perform a PCR reaction in 50 &#181;L volume using DNA polymerase according to the manufacturer&#39;s protocol14.</li>
	<li>Prepare a 1% agarose gel from 0.5 g of agarose, 50 mL of TAE buffer (40 mM Tris Acetate, 1 mM EDTA, pH 8.0), and 3 &#181;L of DNA stain.</li>
	<li>Mix 5 &#181;L aliquot of the PCR-amplified product with 5 &#181;L of RNase/DNase-free water and 2 &#181;L of 6x gel loading dye and analyze by agarose gel electrophoresis. Load a DNA ladder (0.1-10.0 kb) as a reference. Run the gel at 120 V until the dye line has almost reached the bottom of the gel.</li>
	<li>Visualize the DNA fragments using a UV transilluminator imaging system to check for the correct size of DNA15.</li>
	<li>Purify the PCR product using a PCR purification kit according to the manufacturer&#39;s protocol16.</li>
	<li>Digest the PCR product and pSAL expression vector with KpnI and BlpI restriction enzymes in a 15 &#181;L reaction, containing 10 &#181;L of PCR product or plasmid vector (concentration 20-50 ng/&#181;L), 1.5 &#181;L of 10x enzyme buffer, 1 &#181;L of each enzyme, and 1.5 &#181;L of RNase/DNase-free water, at 37 &#176;C for 2 h.</li>
	<li>Add 3 &#181;L of 6x gel loading dye to the reaction mixture and separate the digested fragments on a 1% agarose gel as described in steps 1.3-1.5.</li>
	<li>Purify the DNA fragments using a gel extraction kit according to the manufacturer&#39;s protocol16.</li>
	<li>Ligate the digested PCR product into a digested linearized plasmid vector, pSAL, using T4 DNA ligase and supplemented ligase buffer in a 10 &#181;L reaction containing 3-20 fmol of the digested vector, 9-60 fmol of the digested PCR product, 2 &#181;L of ligase buffer, 1 &#181;L (1 unit) of T4 DNA Ligase, and DNase/RNase-free water. Incubate the ligation reaction at 25 &#176;C for 3 h. 
	NOTE: Ensure that the total DNA content in the reaction mixture is 0.01-0.1 &#181;g.</li>
	<li>Transform E. coli DH5&#945; competent cells with 10 ng of the ligation reaction mixture according to the manufacturer&#39;s protocol17.</li>
	<li>Grow the transformed cells at 37 &#176;C overnight on LB-agar plates supplemented with ampicillin (100 &#181;g/mL).</li>
	<li>Pick 3-4 bacterial colonies from the plate and aseptically transfer each of them into 5 mL of LB media supplemented with ampicillin (100 &#181;g/mL). Grow the cultures overnight at 37 &#176;C in a shaking incubator at 225 rpm.</li>
	<li>Pellet the overnight cultures by centrifugation at 11 x g for 3 min at room temperature.</li>
	<li>Use a purification kit to purify the plasmids according to the manufacturer&#39;s protocol16.</li>
	<li>Verify the sequences of the purified plasmids by DNA sequencing. The plasmid map and the sequence of the resulting construct (pSALv-RS-GFPa1) are shown in Figure 1.</li>
</ol>
2. Large-scale DNA purification.
<ol>
	<li>Transform the plasmid vector pSALv-RS-GFPa1 (3.4 kb) (encoding theophylline riboswitch coupled with GFPa1 reporter gene) or pET28c-F30-2xBroccoli (5.4 kb) (encoding Broccoli aptamer) into E. coli DH5&#945; competent cells according to the manufacturer&#39;s protocol17.</li>
	<li>Grow the transformed cells at 37 &#176;C overnight on LB-agar plates supplemented with ampicillin (100 &#181;g/mL) for cells transformed with pSALv-RS-GFPa1 or kanamycin (50 &#181;g/mL) for cells transformed with pET28c-F30-2x Broccoli.</li>
	<li>Pick 3-4 bacterial colonies from the plate and aseptically transfer each colony into 5 mL of LB media supplemented with appropriate antibiotic (100 &#181;g/mL ampicillin or 50 &#181;g/mL kanamycin). Grow the cultures overnight at 37 &#176;C in a shaking incubator at 225 rpm.</li>
	<li>Use 3 mL of the overnight culture to inoculate into 150 mL of LB supplemented with appropriate antibiotic (100 &#181;g/mL ampicillin or 50 &#181;g/mL kanamycin) and grow the cultures overnight at 37 &#176;C in a shaking incubator at 225 rpm.</li>
	<li>Pellet the cells by centrifugation at &#8805;3400 x g for 10 min at 4 &#176;C.</li>
	<li>Use a purification kit to purify the plasmids according to the manufacturer&#39;s protocol16.</li>
	<li>Elute the DNA with 0.5 mL of pure DNase/RNase-free water. Measure the DNA concentration and prepare 1 mL of DNA stock solutions (100 ng/&#181;L). Store the tubes with DNA at 4 &#176;C until further use.</li>
</ol>
3. Extraction of silk fibroin and preparation of initial materials.
<ol>
	<li>Prepare an aqueous solution of reconstituted silk fibroin (SF) protein from Bombyx mori silkworm cocoons according to the procedure described in detail elsewhere to account for 10% of Silk-LiBr solution18.</li>
	<li>Determine the final concentration of the aqueous SF solution. Pipette 0.5 mL of silk solution to a 60 mm Petri dish, let it dry at 60 &#176;C, and measure the weight of dry silk film. Divide the dry weight by 0.5 mL to calculate the weight per volume percentage.</li>
	<li>Dilute the concentrated silk solution with DNase/RNase-free distilled water by slowly adding water via serological pipette to obtain 1 mg/mL final concentration. Store the solution at 4 &#176;C for future use.</li>
	<li>Prepare fluorescently labeled silk fibroin using an antibody labeling kit. Use 1 mL of 2 mg/mL silk fibroin solution to couple the N-terminal &#945;-amino groups of the protein with an NHS ester-activated derivative dye according to the manufacturer&#39;s protocol19.</li>
	<li>Prepare 50 mL of polyethyleneimine (PEI) aqueous solution with a concentration of 6 mg/mL, adjust pH to 4 with HCl (1 M). Filter the solution through a sterile 0.2 &#181;m membrane. Storage is possible at ambient conditions for months.</li>
	<li>Prepare SiO2 cores. Pipette 300 &#181;L of SiO2 particles into a 2 mL microcentrifuge tube. Wash the microparticles two times with 1 mL of DNase/RNase-free water by centrifugation at 0.2 x g for 1 min.</li>
</ol>
4. Perform Layer-by-Layer deposition of a prime layer, DNA plasmids, and silk layers.
<ol>
	<li>To deposit the PEI prime layer onto the SiO2 microparticles, add 1 mL of PEI solution to the spun down pellet from step 3.6 and agitate the mixture at ambient conditions on a thermomixer at 800 rpm for 15 min. Wash the particles four times with 1 mL of DNase/RNase-free deionized water by centrifugation at 0.2 x g for 1 min.</li>
	<li>To perform deposition of the DNA layer, add 1 mL of the aqueous solution of DNA plasmids from step 2.7 to the PEI-primed microparticles and gently agitate the mixture at 4 &#176;C on a thermomixer at 800 rpm for 15 min. To prepare microcapsules with different DNA loads, adjust the concentration of DNA plasmids from 50-200 ng/&#181;L using DNase/RNase-free distilled water and use 1 mL of these solutions to deposit the DNA. Collect the microparticles by centrifugation at 0.2 x g for 1 min.</li>
	<li>Mark the tubes for DNA plasmids encoding theophylline riboswitch coupled with GFPa1 as ThyRS-GFPa1, and DNA plasmids encoding Broccoli aptamer as BrocApt. 
	NOTE: Keep the microcentrifuge tubes with DNA on ice.</li>
	<li>Carefully remove the supernatant and wash the microparticles four times with 1 mL of DNase/RNase-free distilled water, each time discarding the supernatant after centrifugation at 0.2 x g for 1 min. Perform all experiments at room temperature (RT) unless it is specified otherwise.</li>
	<li>To perform deposition of the silk fibroin layer add 1 mL of the reconstituted aqueous SF solution from step 3.3 to the DNA-adsorbed microparticles, gently vortex and agitate the mixture at 10 &#176;C on the thermomixer at 750 rpm for 15 min. Collect the microparticles by centrifugation at 0.2 x g for 1 min at 4 &#176;C, remove the supernatant, and then wash them once with 1 mL of DNase/RNase-free distilled water. Repeat the centrifugation and discard the supernatant. 
	NOTE: During the experiment, keep the solution of silk on ice to avoid temperature-induced gelation.</li>
	<li>Gradually treat the particles with methanol to induce &#946;-sheet formation in silk protein structure. First, add 0.5 mL of DNase/RNase distilled water, vortex the microcentrifuge tube, then add 0.5 mL of 100% methanol. Gently shake the particles on the thermomixer at 10 &#176;C for 5 min. Collect the particles by centrifugation at 0.2 x g for 1 min. Remove the supernatant.</li>
	<li>Treat the particles with methanol to promote the formation of &#946;-sheets and ensure strong physical adsorption of the silk layer. Add 1 mL of 100% methanol. Gently shake the particles on the thermomixer at 750 rpm for 10 min at 10 &#176;C.</li>
	<li>Collect the particles by centrifugation at 0.2 x g for 1 min at 4 &#176;C and wash them twice with 1 mL of DNase/RNase-free distilled water each time, discarding the supernatant and gently vortexing before the next centrifugation.</li>
	<li>Repeat steps 4.5-4.8 20 times to obtain silk multilayered core-shell structures. For the last deposition step, use fluorescently labeled silk from step 3.4 (Silk-DyLight550, 1 mL).</li>
	<li>Perform the last washing step and keep the microparticles in 1 mL of DNase/RNase-free distilled water at ambient conditions. 
	NOTE: To avoid aggregation of particles during deposition of silk layers, perform a visual inspection of the particles suspension and pipette it up and down using a 1 mL pipette tip to promote homogeneous particle distribution.</li>
	<li>Calculate the number of DNA plasmid copies encapsulated in each microcapsule, NDNA using Equation 1: 
	<img alt="Equation 1" src="/files/ftp_upload/62854/62854eq01.jpg" /> (1) 
	Where N = 6.769 &#215; 1011 - the number of SiO2 cores used for encapsulation. Calculate it from a standard curve for known concentrations of silica particles using serial dilutions and absorption A320 at &#955; = 320 nm; 
	C- initial concentration of DNA used for adsorption 
	V- the volume of DNA used for adsorption 
	0.8- DNA adsorption efficiency on the cores 
	Mw- Molecular weight of DNA plasmid 
	NA- Avogadro&#39;s number (6.022&#160;&#215; 1023)</li>
</ol>
5. Dissolution of cores to obtain silk microcapsules.
<ol>
	<li>Prepare 8% hydrofluoric acid (HF) solution, pH 5.5, by diluting stock solution (48%) with distilled water. Acquire a 50 mL centrifuge tube. Carefully pipette 5 mL of HF and add 25 mL of distilled water to obtain 8% HF solution. 
	CAUTION: HF is a highly corrosive acid and may cause severe burns to the tissues. Extreme caution must be taken during the handling and use of HF for the experiments. Adhere to Standard Operating Procedure (SOP) for the proper usage and handling of HF developed by the organization to avoid undesirable spillage accidents. Do not use glass containers to dilute HF acid. Use the chemical hood to perform this step of the protocol.</li>
	<li>Dissolve SiO2 cores by adding 1.5 mL of 8% HF solution to pelleted core-shell microparticles from step 4.10. Gently vortex and let cores dissolve overnight at ambient conditions with gentle shaking at 450 rpm. 
	NOTE: To avoid spillage of HF, use grafting tape to seal the microcentrifuge tube. Use a chemical hood to perform this step of the protocol.</li>
	<li>Prepare a 2 L glass beaker filled with 2 L of deionized water. Transfer microcapsules solution to dialysis devices (50 kDa MWCO) and dialyze them against deionized water with a repeated change of the water every 3 h for the next 3 days. 
	NOTE: Collect the supernatant during the first three exchanges of water and discard the solution according to the established protocol for hazardous waste materials.</li>
	<li>Use a 1 mL pipette to transfer the suspension from dialysis devices into new 2 mL microcentrifuge tubes to collect the microcapsules. 
	NOTE: Store the aqueous solutions of microcapsules at ambient conditions for several years.</li>
</ol>
6. Imaging of silk fibroin microcapsules using confocal laser scanning microscope (CLSM).
<ol>
	<li>Perform DNA staining using a DNA dye.
	<ol>
		<li>Transfer 300 &#181;L of hollow silk fibroin microcapsules into a fresh 1 mL microcentrifuge tube. Add 500 &#181;L of RNase/DNase-free distilled water.</li>
		<li>Add 5 &#181;L of the DNA staining dye, briefly vortex, and incubate at RT for 2 h protected from light.</li>
		<li>Perform four washing steps by centrifugation at 0.1 x g for 20 min at 4 &#176;C each time, carefully removing 400 &#181;L of the supernatant and replenishing it with 400 &#181;L of RNase/DNase-free distilled water.</li>
	</ol>
	</li>
	<li>Perform the imaging of silk capsules on inverted confocal systems equipped with three major lasers (405 nm, 488 nm, 561 nm) using 100x oil-immersion objective (NA 1.49). Transfer 100 &#181;L of the capsule sample into a single well of 8-well chambered glass slides, allow for capsules to sediment for 20-30 min prior to imaging. 
	NOTE: Dyes are very sensitive to photobleaching. Protect the samples by covering the slides with aluminum foil.</li>
</ol>
7. Estimation of the permeability of hollow microcapsules using molecular weight cut-off (MWCO) method.
<ol>
	<li>Prepare 2 mL each of FITC-labeled dextran fluorophore solutions (20 &#181;M, diH2O) of different Mw (4 kDa, 20 kDa, 40 kDa, 70 kDa, 150 kDa, 250 kDa, 500 kDa, and 2 MDa).</li>
	<li>Pipette 100 &#181;L of the suspension of capsules into a single well of a chambered glass slide. Analyze each microcapsule design (concentration of PEI, loading number of DNA plasmids, the concentration of silk fibroin, and the number of layers) separately.</li>
	<li>To each well, add 300 &#181;L of the specific fluorophore solution starting from the lowest Mw&#160;up to the highest, so each well will correspond to the specific fluorophore solution. Mix by pipetting up and down and let the mixture incubate for 1 h at RT until the diffusion of fluorophore solutions reaches equilibrium.</li>
	<li>Transfer the slide to a confocal laser scanning microscope (CLSM) and image each well using 100x oil-immersion objective at excitation &#955; = 488 nm.
	<ol>
		<li>Identify the area of interest by adjusting the focal plane to make sure that the capsules appear in the form of circles of the largest diameter. This typically happens when viewing the samples closer to the bottom of the well when capsules sediment due to gravity.</li>
		<li>Collect several images of microcapsule samples by moving the slide in XY direction. Capture images to account for up to 100-150 capsules for each sample.</li>
		<li>Use ImageJ software to analyze the permeability of the capsule&#39;s membrane in each Mw fluorophore solution by comparing fluorescence intensities inside and outside of the capsules. For that, draw a region of interest (ROI) in the form of a circle to outline the circumference of the capsule and click Analyze/ Measure to measure the fluorescence intensity inside. Tabulate the data into a spreadsheet. Perform this operation for each microcapsule for a total of 200-300 capsules.</li>
		<li>Assess the outside fluorescence intensity in the same way by outlining the ROI and measuring the intensity away from the capsules. Perform 3-5 measurements for statistical analysis.</li>
		<li>To perform statistical analysis, compare the fluorescence intensities inside and outside of the capsules using paired t-test (p &#60; 0.05).</li>
		<li>Use the conversion Table 2 to estimate the permeability of microcapsules based on the hydrodynamic radii for FITC-Dextran with variable Mw.</li>
	</ol>
	</li>
</ol>
8. In vitro activation of synthetic theophylline riboswitch in silk microcapsules
<ol>
	<li>Prepare 1 mL of theophylline stock solution (100 mM, DMSO). Prepare E. coli S30 extract system for circular DNA by thawing the components on ice for 40 min.</li>
	<li>Obtain a 0.5 mL DNase/RNase-free microcentrifuge tube. Perform in vitro transcription/translation reaction, combine the cell-free components with a sample of microcapsules in the following order (50 &#181;L total volume): S30 premix without amino acids (20 &#181;L); S30 extract, circular (15 &#181;L); complete amino acids mixture (5 &#181;L); hollow microcapsules containing ThyRS-GFPa1 plasmids from step 4.10 (9 &#181;L); and theophylline, 100 mM DMSO (1 &#181;L). 
	NOTE: After adding all components, briefly vortex the tube and collect the sample during brief centrifugation at 0.2 &#215; g for a couple of seconds.</li>
	<li>Incubate the tube at 30 &#176;C for 4 h and check the fluorescence on a plate reader using excitation at &#955; = 488 nm and emission for GFP/FITC filter (510 nm &#177; 20 nm).</li>
	<li>Image the capsules on any LCSM system using 488 nm and 561 nm lasers. Obtain the best quality images using 100x oil immersion objective and 8-well chambered slides.</li>
</ol>
9. In vitro activation of broccoli aptamer in silk microcapsules
<ol>
	<li>Prepare 1 mL of stock solution of DFHBI-1T dye (30 &#181;M, diH2O). Prepare the PURE (protein synthesis using recombinant elements) cell-free system reaction kit by thawing the components on ice for 40 min.</li>
	<li>Obtain a 0.5 mL DNase/RNase-free microcentrifuge tube. Perform in vitro transcription reaction by combining cell-free reaction components with a sample of microcapsules in the following order (50 &#181;L total volume): solution A (20 &#181;L); solution B (15 &#181;L); hollow microcapsules containing BrocApt plasmids from step 4.10 (14 &#181;L); and DFHBI-1T dye (1 &#181;L). 
	NOTE: After adding all components, briefly vortex the tube and collect the sample during brief centrifugation at 0.2 &#215; g for a couple of seconds.</li>
	<li>Incubate the tube at 37 &#176;C for 6 h and check the fluorescence on a plate reader using excitation at &#955;ex = 470 nm and emission at &#955;em = 510 nm &#177; 20 nm.</li>
	<li>Image the capsules on any LCSM system using 488 nm and 561 nm lasers. Obtain the best quality images using 100x oil-immersion objective, and 8-well coverglass chambered slides.</li>
</ol>

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L., Kelley-Loughnane, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+activity+of+DNA-encoded+sensing+elements+through+confinement+in+silk+microcapsules.">Improving the activity of DNA-encoded sensing elements through confinement in silk microcapsules.</a> ACS Applied Materials &amp; Interfaces. 12 (43), 48329-48339 (2020).</li><li>Melnikov, S., Ben-Shem, A., Garreau de Loubresse, N., Jenner, L., Yusupova, G., Yusupov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+inhibition+of+the+eukaryotic+ribosome.">Structural basis for the inhibition of the eukaryotic ribosome.</a> Nature Structural &amp; Molecular Biology. 19 (6), 560-567 (2012).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+layer-by-layer+assembly:+A+tribute+to+ACS+Nano+associate+editor+Helmuth+M&#246;hwald.">The future of layer-by-layer assembly: A tribute to ACS Nano associate editor Helmuth M&#246;hwald.</a> ACS Nano. 13 (6), 6151-6169 (2019).</li><li>Main, K. H. S., Provan, J. I., Haynes, P. J., Wells, G., Hartley, J. A., Pyne, A. L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy-A+tool+for+structural+and+translational+DNA+research.">Atomic force microscopy-A tool for structural and translational DNA research.</a> APL Bioengineering. 5, 031504 (2021).</li><li>Riera, R., Feiner-Gracia, N., Fornaguera, C., Cascante, A., Borr&#243;s, S., Albertazzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+DNA+complexation+state+of+pBAE+polyplexes+in+cells+with+super+resolution+microscopy.">Tracking the DNA complexation state of pBAE polyplexes in cells with super resolution microscopy.</a> Nanoscale. 11 (38), 17869-17877 (2019).</li><li>Bilokapic, S., Strauss, M., Halic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+of+nucleosome+core+particle+interactions+in+trans.">Cryo-EM of nucleosome core particle interactions in trans.</a> Scientific Reports. 8, 7046 (2018).</li><li>Pritchard, E. M., Dennis, P. B., Omenetto, F., Naik, R. R., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+aspects+of+stabilization+of+compounds+in+silk.">Physical and chemical aspects of stabilization of compounds in silk.</a> Biopolymers. 97 (6), 479-498 (2012).</li><li>Fritz, B. R., Jamil, O. K., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+macromolecular+crowding+and+reducing+conditions+for+in+vitro+ribosome+construction.">Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction.</a> Nucleic Acids Research. 43 (9), 4774-4784 (2015).</li><li>Ge, X., Luo, D., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+expression+under+macromolecular+crowding+conditions.">Cell-free protein expression under macromolecular crowding conditions.</a> PLoS One. 6 (12), 28707 (2011).</li><li>Cawte, A. D., Unrau, P. J., Rueda, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+single+RNA+molecules+with+fluorogenic+mango+II+arrays.">Live cell imaging of single RNA molecules with fluorogenic mango II arrays.</a> Nature Communications. 11, 1283 (2020).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+RNA+dynamics+in+live+cells+with+bright+and+stable+fluorescent+RNAs.">Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.</a> Nature Biotechnology. 37 (11), 1287-1293 (2019).</li></ol>

The views and opinions presented herein are those of the authors and do not necessarily represent the views of DoD or its Components

Selectively permeable hydrogel microcapsules loaded with various types of DNA-encoded sensor designs can be prepared following this protocol. One of the distinctive features of the LbL approach is the ability to tailor the complexity of microcapsules during the bottom-up assembly, which usually starts with the adsorption of molecular species on sacrificial templates. By carefully adjusting concentrations of the initial components, pH conditions, and the number of layers, microcapsules with different DNA loading parameter...

The field of synthetic biology offers unique opportunities to develop sensing capabilities by exploiting natural mechanisms evolved by microorganisms to monitor their environment and potential threats. Importantly, these sensing mechanisms are typically linked to a response that protects these microorganisms from harmful exposure, regulating gene expression to mitigate negative effects or prevent intake of toxic materials. There have been significant efforts to engineer these microorganisms to create whole-cell sensors taking advantage of these natural responses but re-directing them to recognize novel targets and/or to produce a measurable signal that can be measured for quantification purposes (typically fluorescence)1,2. Currently, concerns with the use of genetically modified microorganisms (GMOs), especially when releasing in the environment or the human body, due to leakage of whole cells or some of their genetic material, even if encapsulated in a polymer matrix, suggest that alternative ways to exploit these sensing approaches are needed3.
A powerful approach to exploit the benefits of microorganisms-based sensing without the concern for the deployment of GMOs is the use of in vitro transcription/translation (IVTT) systems. From a practical perspective, IVTT systems consist of a mixture containing most of the cell components in an active state that has been &#34;extracted&#34; from cells by different means, including sonication, bead-beating, or others4. The final product of this process is a biochemical reaction mixture already optimized to perform transcription and translation that can be used to test different sensors in an &#34;open vessel&#34; format, without the constraints associated with the use of whole cells (membrane diffusion, transformation efficiency, cell toxicity, etc.). Importantly, different sensor components can be quantitatively added, and their effect studied by different optical and spectrometric techniques, as we have demonstrated5. It has been noticed that the performance of IVTT systems can be inconsistent; however, recent studies have shown approaches to standardize their preparation and characterization, which is of great help when studying their performance in sensor design6. Recently, many examples of IVTT systems using to create paper-based assays through the lyophilization of their components in paper matrices have been demonstrated, including detection of heavy metal ions, drugs, quorum sensing elements, and others7,8,9. An exciting application space for IVTT-based sensors is their use in sensing applications in different types of environments, including soil, water, and the human body. In order to deploy these IVTT systems to these challenging environments, an encapsulation approach need to be implemented to contain the IVTT components and protect them from degradation.
The most common encapsulation approaches for IVTT systems include the use of lipid capsules, micelles, polymersomes, and other tightly enclosed microcontainers10,11,12. One disadvantage of this approach is the need to incorporate either passive or active mechanisms to transport materials in and out of the containers to allow communication with the external environment and provide sensing capabilities. To overcome some of these issues, the study here reports a method that provides a simple yet effective approach to encapsulate the encoding materials for different sensor designs to be expressed in IVTT systems. This approach is based on the use of Layer-by-Layer (LbL) deposition of a biopolymer in the presence of the plasmids of interest to create hollow microcapsules with high porosity, which allows the protected genetic material to interact with the different components of the IVTT of choice. The study demonstrated that encapsulated plasmids could direct transcription and translation when activated within this polymeric matrix, as shown with the response of a plasmid-encoded aptamer and a riboswitch to their corresponding targets. Additionally, this LbL coating protects the plasmids for months without any special storage conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T)</td>
			<td>Lucerna</td>
			<td>DFHBI-1T</td>
			<td></td>
		</tr>
		<tr>
			<td>5x T4 DNA Ligase Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>46300-018</td>
			<td></td>
		</tr>
		<tr>
			<td>6x Blue Gel Loading Dye</td>
			<td>New England BioLabs</td>
			<td>B7021S</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, black circular</td>
			<td>Corning</td>
			<td>3601</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>BioReagent, for molecular biology, low EEO</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>powder or crystals, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>BlpI restriction enzymes</td>
			<td>New England BioLabs</td>
			<td>R0585S</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Disposable Vacuum Filter/Storage Systems</td>
			<td>FisherScientific</td>
			<td>09-761-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide, DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td>ACS reagent, &#8805;99.9%</td>
		</tr>
		<tr>
			<td>DNA Plasmid, pET28c-F30-2x Broccoli (5.4 kb), BrocApt.</td>
			<td>Addgene</td>
			<td>Plasmid #66788</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLightTM550 Antibody Labeling kit (Invitrogen)</td>
			<td>ThermoFisher Scientific</td>
			<td>84530</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli S30 extract system for circular DNA</td>
			<td>Promega</td>
			<td>L1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Conical centrifuge tubes, 15 mL</td>
			<td>FisherScientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Conical centrifuge tubes, 50 mL</td>
			<td></td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Microcentrifuge tubes, 1.5 mL</td>
			<td>FisherScientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrofluoric acid, HF</td>
			<td>Sigma-Aldrich</td>
			<td>695068</td>
			<td>ACS reagent, 48%</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
			<td>mixture of Kanamycin A (main component) and Kanamycin B and C</td>
		</tr>
		<tr>
			<td>KpnI restriction enzymes</td>
			<td>New England BioLabs</td>
			<td>R0142S</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar plate supplemented with 100 &#181;g/mL ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>L5667</td>
			<td>pre-poured agar plates with 100 &#181;g/mL ampicillin</td>
		</tr>
		<tr>
			<td>LB agar plate supplemented with 50 &#181;g/mL kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>L0543</td>
			<td>pre-poured agar plates with 50 &#181;g/mL kanamycin</td>
		</tr>
		<tr>
			<td>LB broth (Lennox grade)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium bromide, LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>ReagentPlus, &#8805;99%</td>
		</tr>
		<tr>
			<td>Max Efficiency DH5-&#945; competent E. coli strain</td>
			<td>ThermoFisher Scientific</td>
			<td>18258012</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>MilliporeSigma</td>
			<td>322415</td>
			<td>anhydrous, 99.8%</td>
		</tr>
		<tr>
			<td>MilliQ-water</td>
			<td>EMD MilliPore</td>
			<td></td>
			<td>Milli-Q Reference Water Purification System</td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride, EDC</td>
			<td>Sigma-Aldrich</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td>average Mw ~25,000</td>
		</tr>
		<tr>
			<td>PURExpress In Vitro Protein Synthesis Kit</td>
			<td>New England BioLabs</td>
			<td>E6800S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAEX II Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>20021</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>&#160;27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-Load 2-Log DNA Ladder (0.1-10.0 kb)</td>
			<td>New England BioLabs</td>
			<td>N0469S</td>
			<td></td>
		</tr>
		<tr>
			<td>SiO&#8322; silica microspheres, 4.0 &#181;m</td>
			<td>Polysciences, Inc.</td>
			<td>24331-15</td>
			<td>10% aqueous solution</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>87724</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate, Na&#8322;CO&#8323;</td>
			<td>Sigma-Aldrich</td>
			<td>222321</td>
			<td>ACS reagent, anhydrous, &#8805;99.5%, powder</td>
		</tr>
		<tr>
			<td>Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis Devices</td>
			<td>FisherScientific</td>
			<td>08-607-008</td>
			<td>Spectrum G235058</td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase (5 U/&#181;L)</td>
			<td>ThermoFisher Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Theophylline</td>
			<td>Sigma-Aldrich</td>
			<td>T1633</td>
			<td>anhydrous, &#8805;99%, powder</td>
		</tr>
		<tr>
			<td>Tris Acetate-EDTA buffer (TAE buffer)</td>
			<td>Sigma-Aldrich</td>
			<td>T6025</td>
			<td>Contains 40 mM Tris-acetate and 1 mM EDTA, pH 8.3.</td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>FisherScientific</td>
			<td>10-977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>ZymoPURE II Plasmid MaxiPrep kit</td>
			<td>ZymoResearch</td>
			<td>D4202</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

Here, the study addresses the functionality of DNA templates encoding different sensor designs (two types of RNA-regulated transcription/translation elements) after encapsulation in silk protein capsules. Microcapsules were prepared via templated Layer-by-Layer (LbL) assembly of the key components: A prime layer, DNA plasmids encoding sensor designs, and silk fibroin biopolymer (Figure 2). Deposition of macromolecules in a layered fashion allows controlling the permeability of the capsule me...

Watch this Scientific Journal Video about Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches at JoVE.com

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

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

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

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

Research

JoVE Journal

Biology

1.3K Views.  UES Inc.. The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

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

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...