November 6, 2019
by Abhilasha "Abby" Dehankar
Edited by Faiz Khan & Elizabeth Jergens
Goal: Our goal is to develop photoswitchable QD-DNA-AuNP composites that will enable us to use QDs for STORM imaging because of their controllable stochastic on/off behavior. QD-DNA-AuNP composites are designed to form by hybridization of complementary azobenzene modified single-stranded DNA (ssDNA) attached to QDs and AuNPs. Formation of QD-azoDNA-AuNP will yield quenching of QD fluorescence via a Forster Resonance Energy Transfer mechanism. The QD-azoDNA-AuNP composites are tailored to undergo photoactivated reversible QD fluorescence quenching. When UV light is applied the azobenzene molecules switch from trans to cis conformation, promoting dissociation of the double-stranded DNA complexes. Thus, QDs are detached from AuNPs, restoring QD fluorescence. Whereas, when visible light is applied the azobenzene switches back to trans conformation, promoting hybridization and reattachment of QD-azoDNA-AuNP composites and quenching of QD fluorescence.
Figure 1. QD-DNA-AuNP conjugate reversible photoswitching mechanism. QD fluorescence quenches in the proximity of AuNPs through a mechanism called “Forster resonance energy transfer” and as a result QD fluorescence is quenched when the DNA is hybridized in our QD-DNA-AuNP conjugates. On shining a UV wavelength light (300-400 nm) on the QD-DNA-AuNP conjugates, the trans-azobenzene isomerizes to its cis form. This destabilizes the DNA actuating its dehybridization which re-establishes the QD fluorescence. In contrast, shining visible light (wavelength>400 nm) reverses the entire process and quenches the QD fluorescence.
Review of progress-to-date:
1. We successfully transferred organic QDs purchased from NN-labs into the aqueous phase. We achieved this transfer by exchanging the non-polar ligands (Octadecylamine) on the QD surface with polar phytochelatin-3 (PC3) peptides. The resulting QDs (PC3-QDs) are water-soluble and terminated with carboxylate (-COOH) and amine (-NH2) groups on their surfaces.
2. We successfully conjugated -NH2 terminated sulfo-dibenzocyclooctyne (sDBCO) to -COOH terminations on the surface of PC3-QDs to form QD-sDBCO conjugates using commonly-employed carbodiimide chemistry. Carbodiimide chemistry forms an amide (-CONH2-) bond between –COOH groups and -NH2 groups. QD-sDBCO conjugation allowed us to employ “click” chemistry to successfully conjugate ssDNA to PC3-QDs through the attached sDBCO group. To implement this approach, we mixed the QD-sDBCO conjugates with azide (-N3) terminated ssDNA to permit strain promoted alkyne-azide cycloaddition via a click reaction, generating QD-ssDNA conjugates.
3. The synthesized QD-ssDNA conjugates demonstrated increased attraction towards the surfaces as compared to the solution. This was most likely because of the free sDBCO groups on the QD surface that were not passivated by an ssDNA. As a result, the QDs separated out of the solution rendering them unavailable for QD-DNA-AuNP composite formation. We successfully solved the problem of QD dispersion by addition of polyethylene glycol (PEG) molecules in the solution through depletion-based stabilization.
4. We successfully conjugated AuNPs (size-15 nm) with thiol-terminated ssDNA by the formation of Au-S dative bond using an established instantaneous, low pH method.
Research Update – Faiz
Recap from last month: QD procure from NN Labs have a hydrophobic character. The first step involves exchanging the hydrophobic octyldecylamine ligand with phytochelatin-3 (PC3) peptides. We were experiencing problems with a successful QD-PC3 transfer. However, we were able to track down the problem to impurities present in the base used in the protocol. Replacing the base solved the problem and last month update concluded with a successful transfer of QD from organic to aqueous medium.
This month work was focused on the second step of attaching the ssDNA molecules to the QD surface.
Conjugation of NH2 terminated sulfo-dibenzocyclooctyne (sDBCO) to -COOH terminations on the surface of PC3-QDs to form QD-sDBCO conjugates using commonly employed carbodiimide chemistry.
Figure 2. Carbodiimide chemistry (EDC: 1-Ethyl-3-(3 dimethylamino propyl)carbodiimide; sNHS: sulfo-N-hydroxysuccinimide
Mixing the QD-sDBCO conjugates with azide (-N3) terminated ssDNA to permit strain promoted alkyne-azide cycloaddition via a click reaction, generating QD-ssDNA conjugates.
Figure 3. Conjugation of Azide terminated DNA using strain promoted alkyne-azide cycloaddition click reaction (sDBCO- sulfo-Dibenzocyclooctyne; ssDNA- single-stranded DNA)
DNA molecules have an azide termination on one end, and a dye Cyanine-5 (Cy5) attached to the other end. Azide moiety is essential for the click reaction with QD-sDBCO, whereas Cy5 at the other end helps to confirm the attachment of DNA molecules to QD.
Figure 4. Fluorescence intensity of QD-DNA conjugate
(QD concentration: 0.376 uM; DNA concentration: 1.88 uM)
From the fluorescence data and calibration curve for ssDNA, I was able to calculate the number of DNA molecules conjugated per QD. On average, there are ~5 DNA molecules attached per QD. The quantity of attached DNA molecules closely matches to number achieved when Abhilasha conducted the QD-DNA conjugation protocol.
Also, Elizabeth was successfully able to conjugate the complementary ssDNA on the gold nanoparticle surface. Hence, future experiments will be focused on the photoswitching studies of the QD – Au nanocomposite.
Research Update – Elizabeth
β1 labeling of U87 cells using DNA cages reversibly attached to secondary antibodies previously showed a large amount of non-specific binding. In order to eliminate this problem, a panel of different blocking buffers was tested. The cells are not labeled with a primary antibody, but are labeled with DNA cages attached to secondary antibodies. None of the blocking buffers tested showed much signal so the condition of 1% BSA was chosen for future experiments.
Figure 5. U87 cells without primary antibodies and with secondary antibodies and DNA cages in 1% BSA solution
After determining the best blocking buffer, an experiment was run that found there was likely an excess of secondary antibodies in the system. In order to fix this, the experiments were run with samples that have been column purified to remove any excess antibodies. The next test had four experimental conditions in total, low and high concentration nanoparticles with 50% Cy3 and 50% NH2 termination and nanoparticles with 50% OH and 50% NH2 termination.
In these experiments the cells are labeled with a primary antibodies, fluorescent secondary antibodies that bind to the primaries, and fluorescent DNA cages that reversibly bind to the secondaries. None of the samples had a significant fluorescent signal from the secondary antibody. Though most of the samples also showed a similar level of fluorescence from the DNA cages except for the high concentration 50% Cy3 and 50% NH2.
Figure 6. U87 cells labeling with primary antibodies, secondary antibodies, and DNA cages made with high concentration 50% Cy3 and 50% NH2.
Nucleus staining with dapi (left), DNA cage staining in fitc (middle), and secondary antibody in tritc (right)
Future steps include optimizing the antibody-DNA conjugation process before trying cell labeling again with DNA cages and with QDots.