oxLink™ and sxLink™ Photocrosslinking Technologies
| Technique Note TN202101 |
| oxLink™ and sxLink™ Products |
Dynamic biomolecular interactions are responsible for many cellular functions. Heterobifunctional photo-crosslinking reagents are time-tested tools for studying these interactions. In general, these reagents are small organic compounds containing one chemical group for anchoring to the biomolecule of interest and another photoactivatable group for covalently crosslinking the interacting partner(s). At CellMosaic, we have developed two types of proprietary photo-crosslinking reagents: the oxLink™ and sxLink™ reagents (Figure 1). These reagents combine the highly efficient carbene-generating phenyldiazirine group with convenient reversible chemical crosslinking and the super hydrophilic AqT™ linker.
Figure 1. General chemical structures of oxLink™ and sxLink™ reagents. A typical regent contains (i) a phenyldiazirine photoactive group that photo-crosslinks the interacting biomolecular partner; (ii) a chemical crosslinking group (thiol or aminooxy) capable of forming a reversible linkage after crosslinking to a biomolecule or a preformed releasable linkage (disulfide bond or oxime) with a chemical crosslinking group; and (iii) a flexible linker for tethering these two functional groups. The releasable linkage can be used for downstream purification, enrichment of crosslinked products, and identification of the crosslinking sites in combination with advanced mass spectrum analysis.
Photo-crosslinking Group
The properties of the photo-crosslinking reagents are important for obtaining reliable crosslinking data. First, the reagent should be stable under the experimental conditions. Second, the photolabile group should possess appropriate photolysis properties. In other words, the photolysis conditions should not interfere with or alter the function of the biomolecules involved. Third, the photo generated reactive intermediates should be highly reactive and should not rearrange to less reactive intermediates. CellMosaic’s oxLink™ and sxLink™ reagents use highly efficient carbene-generating 3-trifluoromethyl-3-phenyldiazirine as a photo-crosslinking group. 3-Trifluoromethyl-3-aryldiazirines photolyze at longer UV wavelength (~360 nm), at which photodamage to biomolecules is minimized. The generated carbene inserts C−H bonds within picoseconds. Because the electron-withdrawing trifuoromethyl group confers stability on the intermediate diazo-isomer, no side products are detected under normal labeling conditions with the diazo-isomer
Chemical Crosslinking Group
CellMosaic’s sxLink™ reagent contains a sulfhydryl-specific pyridyl thiol group, enabling attachment to specific cysteines or free thiols on a biomolecule. If the biomolecule does not have a free thiol group, sxLink™ can also be synthesized with a preformed disulfide bond with a chemical crosslinking group, such as carboxylic acid, for labeling any native biomolecules.
oxLink™ reagent contains an aminooxy group for chemical crosslinking of the biomolecule containing a ketone or aldehyde group. oxLink™ reagent can also be synthesized with a preformed oxime bond with a chemical crosslinking group, such as carboxylic acid, for labeling any native biomolecules. Aminooxy-ketone ligation is orthogonal to peptide chemistry and will allow highly selective solid phase-based separation of the aminooxy-labeled crosslinked products. In particular, the selective purification method will permit the characterization of protein complexes in complex matrices, such as plasma, cellular membranes, and cell lysates where these samples may contain free thiols that interfere with the labeling and purification processes.
Linker
oxLink™ and sxLink™ can be linked via ethylene, ethylene glycol, or superhydrophilic AqT™ linker. AqT™ linkers are novel proprietary biomaterials invented at CellMosaic that are chemically assembled from a class of natural and edible sugar alcohol (SA) compounds with properties by design. As the 3-trifluoromethyl-3-phenyldiazirine group is highly hydrophobic, biomolecules labeled with a 3-trifluoromethyl-3 phenyldiazirine compound using a traditional ethylene and ethylene glycol-type linker tend to aggregate and destabilize the labeled protein. The oxLink™ and sxLink™ reagents with AqT™ linker have greatly increased water solubility, better biocompatibility, and decreased non-specific hydrophobic interactions with other biomolecules. oxLink™ and sxLink™ based photo-crosslinking reagents also allow high loading of phenyldiazirine groups with greatly increased chances of photo-crosslinking.
Figure 2. C18 HPLC analysis of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic Acid (green, not soluble in water), phenyldiazirine oxLink™ (T2A10) (blue, very soluble in water: >27 mg/mL), and phenyldiazirine sxLink™ (T2A14) (red, modest soluble in water: 2.2 mg/mL saturated solution). HPLC method: Buffer A: 0.1% TFA in water, Buffer B: 0.1% TFA in acetonitrile. 5 to 95% B within 12 minutes.
Application and Workflow for oxLink™ and sxLink™
Figure 3 and 4 show the workflow for how a reversible thiol or oxime linker can be used for crosslinking the interacting biomolecuel partner and identifying the crosslinking site or detecting the interaction partner.
Figure 3. sxLink™ workflow using water-soluble phenyldiazirine sxLink™ (T2A14). First, sxLink™ is tethered to a target biomolecule of interest (M1) via the chemical disulfide crosslinker (thiol exchange). The progression of labeling can be followed by UV analysis of the released chromophore 2-thiolpyridone at 343 nm. The modified biomolecule (M1) is then reconstituted into its native complex with biomolecule M2. A phenyldiazirine group is then activated by UV irradiation, resulting in a crosslink with the neighboring biomolecule (M2). Finally, the disulfide bond connecting the two biomolecules is cleaved by a reducing reagent, revealing the biomolecule interaction by transfer of the free thiol to M2.
Figure 4. oxLink™ workflow using water-soluble phenyldiazirine oxLink™ (T2A10). First, oxLink™ is tethered to a target biomolecule of interest (M1) with an artificially introduced ketone or aldehyde group via the aminooxy functional group. The resulting oxime bond can be hydrolyzed, releasing the photo-crosslinked biomolecule (M2) with a free aminooxy group. M2 can then react with any aldehyde- or ketone-containing molecule, such as solid phase, biotin, or fluorescent dye, for further purification, detection, and identification.
Patent and license restrictions
oxLink™ and sxLink™ crosslinking reagents are covered under US patent number 8907079B2, 9511150B2, US14/965,699, and equivalent patents and patent applications in other countries in the name of CellMosaic, Inc. The purchase of oxLink™ and sxLink™ products includes a limited license to use the AqT™ products for internal research and development but not for any commercial purposes. Commercial use shall include:
1. Sale, lease, license or other transfer of the material or any material derived or produced from it.
2. Sale, lease, license or other grant of rights to use this material or any material derived or produced from it.
3. Use of this material to perform services for a fee for third parties, including contract research and drug screening.
If you are interested in using oxLink™ and sxLink™ reagents for commercial usage, please contact us for more information.
References
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