Department of Chemistry, Stony Brook University
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Rubino, F. A., Oum, Y. H., Rajaram, L., Chu, Y., Carrico, I. S. Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry. J. Vis. Exp. (66), e4246, doi:10.3791/4246 (2012).
The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein – strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.
Refer to Table 1 for preparation of all media, buffers and solutions referenced in this protocol.
1. Production of Aha-Labeled Adenovirus
2. Production of Azido-Sugar-Labeled Adenovirus
3. Purification of Azide-Labeled Adenoviral Particles
4. Ligation of Modified Adenoviral Particles with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)
5. Ligation of Modified Adenoviral Particles with Alkyne Probes Using Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) in Deoxygenated Atmosphere
6. Purification and Storage of Labeled Virus Particles
7. SDS-PAGE of Labeled Adenovirus & Fluorescent Gel Scanning
8. Representative Results
The observed results of SDS-PAGE & fluorescent gel scanning of TAMRA-conjugated Ad5 are shown in Figure 3. Although these fluorescent gel scans were performed on samples modified via CuAAC, results should be comparable when probes are ligated via SPAAC. These gel scans indicate that Aha labeling results in modification of the three Ad5 capsid proteins (Hexon, Penton, and Fiber), while sugar labeling modifies only the Fiber capsid protein. Furthermore, an increase in Aha concentration (4 mM vs. 32 mM) results in a greater degree of Aha incorporation, but it also has been shown to decrease infectivity. Prior results7 indicate that for Ad5 produced in 4mM Aha, roughly 5% of exposed methionine sites are TAMRA-modified, with this number increasing to 10% when 32 mM Aha is used. This corresponds to approximately 280 and 510, respectively, dye-labeled sites per Ad5 particle. Analysis of sugar-labeling1 has indicated that roughly 22 of the 36 glycosylation sites on Ad5 are occupied by an azido sugar and subsequently labeled with TAMRA.
If ten tissue culture plates are used to produce azide-modified Ad5 as described in this protocol, 200 - 300 μl of azide-modified virus should be obtained from the white band collected in step 3.6, with a titer of approximately 1.5 × 1012 particles / ml. A common difficulty of this procedure is the absence of a viral band at this purification step, which can be attributed to low viral titer. This is generally a result of infecting cells which are either over- or under-confluent, or by washing away cells - which become loosened from the plate after infection - while rinsing during step 1.
Figure 1. Protocol overview. Azide-containing Aha or GalNAz is first incorporated into adenovirus particles. Ligation with a fluorescent alkyne probe is then performed via "click" chemistry. Finally, SDS-PAGE is used to demonstrate ligation of the fluorescent probe.
Figure 2. Ligation of alkyne probe onto azide-modified virus via (a) SPAAC, (b) CuAAC under de-oxygenated and (c) oxygenated atmosphere.
Figure 3. Fluorescent scans of SDS-PAGE of azide-labeled adenovirus type 5 (Ad5) after CuAAC conjugation with TAMRA-Alkyne. (a) Aha-labeled Ad5, using two concentrations of Aha. Labeling appears to occur on adenovirus Hexon, Penton and Fiber capsid proteins, with the extent of labeling increasing with increased Aha concentration. (b) Ac4GalNAz-labeled adenovirus appears to be labeled only on the Fiber capsid protein.
|Ad5 storage buffer||5 mM Tris-HCl buffer (pH 8.0) containing 0.5 mM MgCl2, 50 mM NaCl, 25% (v/v) glycerol, and 0.05% (w/v) Bovine Serum Albumin (BSA)||Store at -20 °C|
|Ad5 infection buffer||2% (v/v) bovine calf serum, 1× TC solution, Ad5 in storage buffer (volume determined using an MOI of 10 PFU / cell and an infectivity index of 1:20). Bring the solution to the final volume using TD buffer||Ad5 concentration may be determined via UV absorption.|
|TC solution (200×)||136 mM CaCl2, 100 mM MgCl2||Store at 4 °C|
|TD buffer||137 mM NaCl, 5 mM KCl, 0.07 mM Na2HPO4, and 25 mM Tris-HCl, pH 7.5||Store at 4 °C|
|HEK 293 cell growth medium||Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) bovine calf serum (BCS) and 1% (v/v) Pen Strep||Store at 4 °C|
|Cys stock solution (100×)||DMEM (-Met / -Cys) supplemented with 200 mM L-Cysteine||Prepare fresh and filter with a 0.2-μm sterile syringe filter before use.|
|Aha stock solution (25×)||DMEM (-Met / -Cys) supplemented with 100 mM L-Azidohomoalanine||Prepare fresh and filter with a 0.2-μm sterile syringe filter before use.|
|Aha labeling medium||DMEM (-Met / -Cys) supplemented with 10% BCS, Aha stock solution (1×), 2 mM L-Cysteine||Prepare fresh before use.
This corresponds to a 4 mM concentration of Aha, although different concentrations may be used (see Representative Results).
|Met stock solution (25×)||DMEM (-Met / -Cys) supplemented with 100 mM L-Methionine||Prepare fresh and filter with a 0.2-μm sterile syringe filter before use.|
|Met control medium||DMEM (-Met / -Cys) supplemented with 10% BCS, Met stock solution (1×), 2 mM L-Cysteine||Prepare fresh before use.|
|Ac4GalNAz stock solution (1,000×)||50 mM peracetylated N-azidoacetylgalactosamine (Ac4GalNAz) in methanol||Store at 4 °C. Ac4GalNAz is a metabolic precursor to GlcNAz.|
|Azido-sugar labeling medium||100 μl Ac4GalNAz stock solution, 100 ml HEK 293 cell growth medium||Allow methanol to evaporate prior to adding HEK 293 cell growth medium.|
|Cesium chloride solutions||CsCl dissolved in TD buffer:
1.25 g/ml (18.08 g / 50 ml H2O)
1.35 g/ml (25.60 g / 50 ml H2O)
1.40 g/ml (31.00 g / 50 ml H2O)
|Density gradients for ultracentrifugation|
|Virus storage buffer||Phosphate buffered saline (PBS), pH 7.2, containing 0.5 mM CaCl2, 0.9 mM MgCl2, and 10% (v/v) glycerol||Store at -20 °C|
|TAMRA-BCN (SPAAC) labeling solution||TAMRA-BCN in DMSO:
||Strain-promoted alkyne probe
For SDS-PAGE, a viral titer of approximately 1012 particles / ml is suggested
|TAMRA-Alkyne (CuAAC) labeling solution||50 μl of azide-modified Ad5 in virus storage buffer (NAd5 determined as above), 1.5 mM bathophenanthroline disulfonate (i.e. 1.5 × final CuBr concentration), TAMRA-Alkyne.
TAMRA-Alkyne molarity (in M) is determined using:
cAlk = 2 × 106 × (NAd5 / NAvo)
(so that the reaction mixture prepared in Step 5 contains 100 alkyne probes per virion)
|Tris buffer is known to be a competitive and inhibitory ligand of copper.8|
|Laemmli sample buffer (2×)||125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, and 0.004% (w/v) bromophenol blue|
|Running buffer||187 mM glycine, 19 mM Tris-HCl, 3.5 mM SDS in H2O||Store at 4 °C|
Table 1. Preparation of the buffers and solutions required by this protocol.
The development of chemoselective and bioorthogonal click reactions, including those of azides, is a rapidly-evolving area of research, and subsequently there is a growing number of these reactions to choose from for bioconjugation applications. We have restricted the scope of this protocol to include only two methods which were chosen because of their usefulness in our own lab and commercial availability of all reagents.
In the first such route - strain-promoted azide-alkyne cycloaddition (SPAAC) - the alkyne is contained within a cyclooctyne ring (Figure 2a). This recently-developed method can be conducted under oxygenated atmosphere and without the need for a copper catalyst.9,10 Until recently, one drawback of this method was the laborious synthesis and reduced commercial availability of these strained alkynes compared with the terminal alkynes used in copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC); however, a growing library of these probes is now commercially available (see table of reagents). For these reasons, we have chosen SPAAC as the method of choice in this protocol.
Also described in this protocol is the ligation of azide-modified adenoviral particles via CuAAC in deoxygenated atmosphere using bathophenanthroline disulfonic acid disodium salt (BDA) as a chelating agent (Figure 2b). This method is convenient in that the BDA catalyst is both commercially available and inexpensive. Furthermore, reported yields are generally highest of the available methods, and complications arising from the generation of reactive oxygen species (cf. oxygenated CuAAC, below) in solution are avoided.8 However, the copper(I) catalyst is known to be cytotoxic,11 and the specialized equipment required to implement this procedure may deter some groups from using it. Nonetheless, we find this method efficient and useful for adenovirus modification.
A third useful method of CuAAC has been recently developed to work under oxygenated atmospheric conditions (Figure 2c).8 This method enjoys the same benefits as the BDA-chelated approach but uses instead THPTA as a chelating agent to accommodate atmospheric oxygen without oxidative destruction of the substrate. One small drawback of this approach is the current lack of commercial availability of THPTA, although straightforward syntheses of this chelating agent have been reported.8 Although we have not performed this ligation method on our own system, THPTA / CuAAC would be expected to produce comparable results to BDA / CuAAC, with the added convenience of oxygenated atmosphere tolerance.
Despite intentionally developing this protocol around commercially available materials, we still wish to recognize the cost advantages and increased flexibility of synthesizing the azides and alkyne probes used herein. Azidohomoalanine can be prepared in as little as three days and for significantly less than commercial sources.6,12 Reported strained alkyne preparations are more difficult11 but afford greater freedom of probe selection.
No conflicts of interest declared.
We would like to acknowledge the NSF for funding (CBET- 0846259).
|Adenovirus type 5 (Ad5) containing a GFP transgene||BCBC||391|
|Human Embryonic Kidney (HEK 293) cells||ATCC||CRL-1573|
|Dulbecco’s Modified Eagle Medium (DMEM), High Glucose||Invitrogen||11965-092|
|Dulbecco’s Modified Eagle Medium, no Methionine, no Cysteine (DMEM -Met / -Cys), High Glucose||Invitrogen||21013-024|
|Bovine Calf Serum||Invitrogen||16170-078|
|Penicillin - Streptomycin 100× Solution (Pen Strep)||Invitrogen||15140-122|
|0.5% Trypsin-EDTA (10×)||Invitrogen||15400-054|
|100 mm Cell Culture Dish, tissue-culture treated polystyrene||BD Falcon||353003|
|Cell culture CO2 incubator|
|Sterile syringe filter (0.2 μm, cellulose acetate)||VWR||28145-477 (NA), 514-0061 (Europe)|
|Avanti J-E centrifuge||Beckman Coulter||369001|
|Conical tubes (50 ml, sterile)||BD Falcon||352098|
|Tube, Thinwall, Ultra-Clear, 13.2 ml, 14 x 89 mm||Beckman||344059|
|Ultracentrifuge equipped with an SW 41 and SW 60 rotor||Beckman|
|Eppendorf Biopur Safe-Lock Tubes, 1.5 ml||Eppendorf||0030 121.589|
|Centri-Sep gel filtration spin columns||Princeton Separations||CS-901|
|Sterile needle, 18 gauge|
|Nitrogen glove bag (if deoxygenated CuAAC is to be performed)|
|Fluorescent gel scanner|
|Ready Gel Tris-HCl Gel||Bio-Rad||161-1105|
|Peracetylated N-azidoacetylgalactosamine (Ac4GalNAz)||Invitrogen||C33365|
|Bathophenanthroline disulfonic acid (BDA) disodium salt||MP Biomedicals||0215011201|
|Dimethyl sulfoxide (DMSO)|
|Tris base (tris(hydroxymethyl)aminomethane)|
|Disodium Phosphate (Na2HPO4)|
|Phosphate buffered saline (PBS)|
|Bovine Serum Albumin|
|SDS (sodium dodecyl sulfate)|
|Cesium Chloride (CsCl)|
|Copper(I) Bromide (CuBr)|
|Calcium Chloride (CaCl2)|
|Potassium Chloride (KCl)|
|Magnesium Chloride (MgCl2)|
|Sodium Chloride (NaCl)|
|Alkyne probe for CuAAC*|
|Strained alkyne probe for SPAAC*|
|TAMRA DIBO Alkyne||Invitrogen||C10410|
* Notable vendors of click chemistry reagents and kits include Invitrogen, Jena Biosciences, Berry Associates, Sigma-Aldrich, Glen Research, Click Chemistry Tools, and Baseclick. A variety of alkyne dyes and targeting ligands can be found in these vendors’ catalogs.