Experimental Update - September

September 11, 2019

by Abhilasha "Abby" Dehankar

QD-DNA conjugation experiments

 

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.

 

Monthly Report

QD-DNA conjugation experimental trials - Faiz

In previous month’s report, we successfully characterized the melting point of new DNA sequence. The new DNA sequence has cis-melting point below room temperature and a trans-melting point well above room temperature, which satisfy requirements for an efficient photoswitch. The next step involves conjugating this new DNA sequence to QD. This month’s update includes brief overview of the conjugation experiments.

Conjugation of ssDNA to QD involves the following steps:

  1. Organic QDs are provided by NN Labs in toluene. For the conjugation experiment, we require the QDs to be water-soluble. Hence, the first step includes the replacement of non-polar ligands (Octadecylamine) on the QD surface with polar phytochelatin-3 (PC3) peptides. The resulting QD-PC3 are water-soluble, having both carboxylate (-COOH) and amino groups (-NH2) on their surface.

Figure 2. Phytochelatin-3 (PC3) coated compact QDs

 

  1. 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 3. Carbodiimide chemistry (EDC: 1-Ethyl-3-(3 dimethylamino propyl)carbodiimide; sNHS: sulfo-N-hydroxysuccinimide

 

 

  1. 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 4. 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 5. Fluorescence intensity of QD-DNA conjugates, QD concentration: 12 nM

 

After completion of conjugation process, fluorescence of QD-DNA conjugate was carried out. The peak at 555 nm indicates QD fluorescence and peak at 670 nm indicates for the fluorescence intensity of Cy5 terminated DNA molecules. As evident from the figure 5, the fluorescence peak for DNA is negligible relative to QD peak. This indicates that the click reaction was not successful, leading to a low degree of QD-DNA conjugation. Presently, we are working to solve and troubleshoot the process for a better DNA conjugation to the QD surface.

 

Issues with the antibody - Elizabeth

We have been using NIH3T3 cells in the immunocytochemistry staining experiment. We have recently realized that NIH3T3 is a mouse line and the primary antibody we have been using is also a mouse antibody. That's to say, when we add the secondary antibody, which is goat anti-mouse, the secondary antibody is of high chance to bind non-specifically to the cells. This has been proved to be true with our negative control where we add only the secondary antibody to the NIH3T3 cells and saw high signal from nonspecific binding. 

We are now switching to HepG2 cells as the model cell line. They are human cells and should work well with the current primary and secondary antibodies we have. We are doing rigorous control experiments to confirm that we get true staining with our system.