The overall goal of this procedure is to demonstrate a flexible method of re-engineering viral surfaces via click chemistry. In the first step of this procedure, human embryonic kidney cells are infected with type five adenovirus and incubated in the presence of either the unnatural amino acid acid or homo alanine or the aceto sugar parasite N acetoacetyl glucosamine. After two days, the azide labeled adenovirus particles are harvested and purified.
Next, the chemo specific Azi Aine cyclo edition is used to ligate fluorescent probes to the surface of these particles. As explained in the written protocol, this is performed using one of two possible ligation methods, strain promoted or copper. One catalyzed azid ine cyclo edition.
In therapeutic applications, these attachments can include polypeptides polymers or small molecules. In this experiment, a fluorescent probe is used so that ligation of the probe onto capsid proteins can be demonstrated via SDS page. There are significant interest in developing viruses as gene delivery agents, particularly in the context of therapeutics, gene delivery, oncotic therapy, vaccine technology.
In that context, tuning the virus host interface is particularly important in terms of getting the appropriate properties for that therapeutic tissue targeting immune stimulation. A number of other factors are important in terms of tuning the host virus interface. In that context, we've developed a two-step labeling protocol.
The first step is the metabolic introduction of unnatural amino acid or unnatural sugar. This installs an anchor point that we can then further elaborate with very, very highly selective chemistries that we can then append those viruses with essentially any functionality we'd like to without significantly perturbing the virus, infectivity, or overall fitness, which is a major bar, a major hurdle to overcome. After sterilizing the hood and lab equipment, prepare 11 100 millimeter culture dishes of HEK 2 93 cells maintained in HEK growth medium at 37 degrees Celsius.
After the cell culture becomes 80 to 90%confluent, remove the growth medium from one of the dishes and wash once with TD buffer. Loosen the cells with one milliliter of trypsin EDTA and incubate for one minute. Add nine milliliters of growth medium and count the number of harvested cells in this dish.
Using a hemo cytometer, calculate the number of adenovirus particles required for infection for each of the 10 remaining culture dishes. Slowly remove the growth medium, then wash with five milliliters of TD buffer. Add one milliliter of infection buffer and incubate for one hour during this period.
Gently shake the dishes side to side every 15 minutes after infection. Carefully add nine milliliters of HEK growth, medium to each dish, and incubate for another 18 hours. From concentrated stock solutions of a HA in Sistine.
Prepare the a HA labeling medium according to the written protocol. Be sure to first filter these stock solutions with a 0.2 micrometer style syringe filter. Sterilize the methionine stock solution for the control in the same way carefully remove the growth medium and wash each culture dish with five milliliters of TD buffer.
Replace this with 10 milliliters of a HA labeling medium and incubate the infected cells for another six hours. The viral capsid proteins will now be translated in the presence of azo homo alanine and absence of methionine. Carefully replace the labeling medium with 10 milliliters of fresh growth, medium, and incubate for another 18 hours.
After completion of this step, proceed to the purification procedure in step three of the video. Alternatively, azi tags can be introduced via azi containing sugars instead of the unnatural amino acid a HA.In this protocol, parasite related n acetoacetyl galacto a metabolic precursor to UDPN Acetoacetyl. Glucosamine is used, grown in the presence of this aceto sugar.
Type five adenovirus will present azi groups only on the fiber to perform sugar labeling. First follow step one of the video until the onscreen prompt. This corresponds to step 1.5 in the written protocol.
Prepare the sugar labeling solution by first pipetting 100 microliters of ATO sugar stock solution into Falcon tubes and allowing the methanol to evaporate. Next, add 100 milliliters of HEK growth medium. Cover the cells in each dish with 10 milliliters of this sugar labeling medium and incubate at 37 degrees Celsius for 44 hours.
Proceed to step three for purification. Transfer the media along with the infected cells from the culture dish to two conical tubes and centrifuge at 2000 G for 10 minutes. At four degrees.
Remove the supernatant and resuspend each pellet in four milliliters of TD buffer using liquid nitrogen in a 37 degree bath. Perform three freeze thaw cycles to lyce the infected cells. Mix the suspension with a vortex between each cycle.
Centrifuge the thaw suspension at 2000 G for 10 minutes to precipitate the cell debris as the adenovirus used for infection in step one, contained A GFP transgene fluorescent activity at this step confirms transduction and expression of viral DNA. Carefully prepare a cesium chloride density gradient from 1.25 grams per milliliter to 1.4 grams per milliliter. Pipee the lysed cell super natin onto the density gradient and centrifuge at 126, 000 G for one hour at 15 degrees Celsius.
After centrifugation, a thick wide band should be present in the centrifuge tube. This is the azi modified virus. Using a star L needle puncture the bottom of the tube so that the content strain dropwise carefully collect this virus.
Band of the two ligation methods demonstrated the strain. Promoted route involves simpler reaction conditions, but has the drawback of a more limited selection of commercially available probes to perform strain promoted ligation. Mix 50 microliters of azi modified virus in virus storage buffer with 0.25 microliters of Tamara BCN labeling solution mixed by tapping the bottom of the tube cover with foil and rotate overnight at room temperature.
Purify the click virus using centris sub gel filtration. Spin columns equilibrating with virus storage buffer. The Deoxygenated copper one catalyzed cyclo edition enjoys greater availability of probes, but has the drawback of requiring specialized equipment to maintain a deoxygenated atmosphere.
To perform copper catalyzed ligation. First, deoxy oxygenate all reaction materials and solutions in a nitrogen glove bag for six hours After this period, prepare the copper one catalyst stock solution in the glove bag to initiate the reaction. Add one microliter of this copper stock solution to a 50 microliter solution of virus probe and chelating agent.
Allow the labeling reaction to proceed overnight in the glove bag. Purify the acept filtration as in step four. After boiling, purified, clicked virus and laly sample buffer for 10 minutes.
Load the wells with the clicked virus and controls and perform the electrophoresis. Keep the tank covered with foil to protect the fluorescent probe from light. After one hour, use a fluorescent gel scanner to scan the gel.
Note that intact or non-I labeled viruses should not displace significant fluorescent labeling for a HA labeling the viruses produced in the higher concentration of a HA demonstrate greater fluorescent labeling due to a higher degree of a HA incorporation. Also note that the sugar labeled virus expresses azi tags only in the fiber capsid protein Starting from 80%Confluence cells account for two days for infection in azi labeling, five hours for harvesting and purification of labeled virus one night for the click reaction, and two hours for SDS page analysis for a total of three and a half days. In summary, two types of azi labeling methods and two azid alkin click chemistry reactions have been demonstrated.
Your application and resources should determine which method or methods are best suited for your purpose. Although all materials in this protocol are commercially available, we have found that synthesizing a HA aceto sugars and INE probes is significantly more cost effective and affords greater flexibility in virus surface modifications.
