July 16, 2019
by Abhilasha "Abby" Dehankar
Blog Author: Faiz Khan
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 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.
Referring back to monthly report of May, Abhilasha successfully demonstrated formation of QD-DNA-AuNP composites, with FRET based quenching and other thermal actuation based experiments. This month’s report is an attempt to reproduce the results using a 12 mixed base pair DNA sequence conjugated to QD with its complementary version attached to Au.
QD-DNA-AuNP composite or mixture of complementary QD-ssDNA and AuNP-ssDNA composites (C) results in reduced QD fluorescence. The decrease in fluorescence of C is generally referred in comparison to free QD-ssDNA conjugates (Q) or a mixture of non-complementary QD-ssDNA conjugates and AuNP-ssDNA conjugates (NC) because of the absence of FRET-based interaction of QDs with AuNP in the latter two. Therefore, we compared the fluorescence of Q, NC, and C to prove the success of QD-DNA-AuNP composite formation. For the experiment, we prepared the following three samples:
1. Q: QD-ssDNA conjugates dissolved in 2 wt% polyethylene glycol, 20 kDa (PEG20K) in PBS at the final concentration of 3 nM.
2. NC: QD-ssDNA conjugates and non-complementary AuNP-ssDNA conjugates (both dissolved in PEG20K in PBS) mixed at the ratio of 2:1 with final QD concentration of 3 nM.
3. C: QD-ssDNA conjugates and complementary AuNP-ssDNA conjugates (both dissolved in PEG20K in PBS) mixed at the ratio of 2:1 with final QD concentration of 3 nM
We observed a decrease in the fluorescence of QDs (Figure 2) in sample C as compared to both Q (~90% decrease) and NC, clearly indicating the successful formation of QD-DNA-AuNP composite. Interestingly, despite the absence of FRET-based AuNP interactions in the NC sample, we observed a significant decrease of QD fluorescence in NC as compared to Q. We performed multiple replicates of the experiment above to ensure the reproducibility of results. Figure 3 displays the results of all the replicates in the form of a bar graph where each bar represents the normalized fluorescence intensity at 560 nm of that sample.