March 31, 2019
by Abhilasha "Abby" Dehankar
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.
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 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. We used a DNA origami platform to test the functionality of the ssDNA-QD and ssDNA-AuNP conjugates. For this, we mixed the nanoparticle (NP) – ssDNA conjugates with DNA origami hinges containing corresponding complementary ssDNA sequences only at a specific location on the hinge arm. Successful NP binding was observed at the designated spot on the hinge arm, indicating that our ssDNA-NP conjugates are functional.
4. Previous post reported our 2nd attempt at the synthesis of QD-DNA-AuNP conjugates using AuNP-ssDNA conjugates with high ssDNA labeling density formed by a new method. The success of composite formation was analyzed using gel electrophoresis, fluorescence, and optical imaging and indicated successful formation. However, visual confirmation with electron microscopy was yet to be undertaken.
In this report, we will shift the gears back to our attempts at forming QD-DNA-AuNP composites. In our previous report (http://qstorm.org/node/566), we reported that the gel electrophoretic separation of our complementary QD-ssDNA and AuNP-ssDNA conjugate mix indicated successful formation of QD-DNA-AuNP composite. However, we still needed to image the QD-DNA-AuNP dimers in electron microscopy to affirm our conclusion from gel electrophoresis results. Unfortunately, although the gel results with my previous batch of QD-ssDNA conjugates were pretty reproducible, I could not reproduce the same gel results with new QD-ssDNA batches. On closer investigation, we observed that the new QD-ssDNA conjugates did not prefer staying dispersed in the aqueous solution. Instead, all QD-ssDNA conjugates in the solution separated on the walls of the centrifuge tube (Fig 2, right). As a result, QD-ssDNA conjugates were unavailable in the solution for binding with the complementary AuNP-ssDNA conjugates. This explained the irreproducibility of our gel results. Further, this phenomenon also displayed concentration dependence. For example, the QD-ssDNA conjugates stopped separating out of the solution for concentrations higher than that required to coat a layer of QD-ssDNA conjugate on the surface of centrifuge tubes. Interestingly, throughout this process the QD-ssDNA conjugates did not lose their fluorescence.
Since it is necessary for both QD-ssDNA and AuNP-ssDNA conjugates to be dispersed in solution for successful hybridization and composite formation, our first task now was to restrict the separation of QD-ssDNA conjugates on the walls of centrifuge tubes. For this, we first decided to test QD-PC3, our bare QDs coated with phytochelatin-3 (PC3) for aqueous solubilization, for stickiness towards the walls of centrifuge tubes. We saw that QD-PC3 did not stick to the sides of the tube at all (Fig 2, left), indicating the stickiness of our QD-ssDNA conjugates arose from the modifications introduced on QD surface during QD-ssDNA conjugation. Additionally, we performed all the experiments in centrifuge tubes with low-affinity towards ssDNA, to ensure that the stickiness was not a result of ssDNA’s charge interaction with the centrifuge tubes. With low ssDNA labeling density (~1 ssDNA/QD) in our QD-ssDNA conjugates and use of low ssDNA-affinity centrifuge tubes, we inferred that the stickiness of our particles was most likely the result of unreacted sulfo-DBCO on the surface of our QDs.
Now that we knew the source of QD-ssDNA conjugate stickiness, we started digging through the literature to investigate the possible ways to prevent it. One obvious choice to avoid stickiness was to cap the exposed sulfo-DBCO on QD-ssDNA surface with a molecule to hinder its interaction with the wall. Since covalent capping is generally limited by the efficiency of employed chemistry, we decided to opt for capping molecules with non-covalent interactions, such as, hydrogen bonds, electrostatic interactions, etc. Biomolecules, such as, streptavidin, bovine serum albumin, and casein, are known to interact non-covalently with nanoparticle surface groups and providing colloidal stability. However, most of these molecules are bulky and could also cloud the ssDNA during interactions hindering the QD-ssDNA conjugates from binding its complementary AuNP-ssDNA conjugates. Although there is a possibility the ssDNA on QD does not get obstructed, we needed a better alternative.
Another, less-known method of improving colloidal stabilization is through depletion-based stabilization. According to depletion stabilization, addition of free, non-interacting, high molecular weight (MW) polymers (~10,000-20,000), such as polyethylene glycol (PEG) to NP solution stabilizes the NP dispersion at certain concentrations, by creating a metastable low-energy state in the dispersed state of the NPs. Since the stabilization provided by this method is not based on interaction of PEG molecules with NP surface, the accessibility of NP surface and therefore the ssDNA on QD surface remains preserved in this method. A recent publication studied depletion-based stabilization of AuNPs in high salt solutions by addition of PEG molecules of varying size and concentration and reported optimal stabilization at 2-5% wt and 20,000 MW PEG. Therefore, we decided to try dispersing our QD-ssDNA conjugates in these PEG solutions and it worked! We observed that dispersing QD-ssDNA conjugates in both 2 and 5% 20k PEG, stabilized them in the solution and completely eliminated their separation on the walls of centrifuge tubes. The dispersion was also found to be stable for long periods (up to 24-48 hours). The addition of high MW also increased the viscosity of QD-ssDNA conjugates, that might not be desirable for in-vivo applications and therefore we decided to limit future experiments to minimum 2% PEG concentrations. Although depletion-based stabilization seems to have temporarily resolved our dispersion issues, we need to confirm that it does not hinder the hybridization of ssDNA on QD surface that would be included in our next month’s report.