January 15, 2019
by Abhilasha "Abby" Dehankar
Goal: Our goal was to test the formation of the Quantum dot – Deoxyribonucleic Acid – Gold nanoparticle (QD-DNA-AuNP) composites. QD-DNA-AuNP composites are designed to form by hybridization of complementary single-stranded DNA (ssDNA) modified QDs and AuNPs. Here, we worked with DNA duplex that was devoid of any azobenzene modification. Formation of QD-DNA-AuNP will yield quenching of QD fluorescence via a Forster Resonance Energy Transfer mechanism. This study will help us quantify the decrease in QD fluorescence on stable QD-DNA-AuNP composites formation to subsequently tune the DNA sequence for maximum quenching in thermally stable QD-DNA-AuNP composites at ambient conditions.
The ultimate goal is to employ azobenzene modified ssDNA to form QD-azoDNA-AuNP composites that would undergo photoactivated reversible QD fluorescence quenching (Fig 1). When UV light is applied the azobenzene molecules switch from a trans to cis conformation, promoting dissociation of the dsDNA complexes. Thus, QDs are detached from AuNPs, restoring QD fluorescence. These photoswitchable QD-DNA-AuNP conjugates will allow us to employ QDs for STORM imaging because of their predicted and controllable stochastic on/off behavior.
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. The previous post reported the comparison of different purification strategies for ssDNA-QD conjugate purification after synthesis and the first attempt of forming QD-DNA-AuNP composites. The QD-DNA-AuNP composite synthesis was unsuccessful, and we discussed the possible reasons for this failure.
This report will include the recent progress in our attempts to synthesize QD-DNA-AuNP composites. As mentioned in the last report, the most likely reason for our failure in synthesizing the QD-DNA-AuNP composites is the lack of sufficient amount or density of complementary ssDNA on AuNP surface. Although many methods are currently available in the literature for increasing the ssDNA labeling density on AuNPs, we decided to use a recent instantaneous, and low pH assisted ssDNA-AuNP conjugation method that promises an increase of ~39% in the ssDNA labeling density as compared to currently implemented salt-aging method for a wide range of AuNP sizes. Next, we synthesized the QD-DNA-AuNP composites using the ssDNA-AuNP conjugates by the new low pH method. Briefly, 15 nm AuNPs conjugated to PolyA ssDNA (PolyA= 20 consecutive Adenosines) in PBS were mixed with green QDs conjugated to PolyT ssDNA (PolyA= 16 consecutive Thymines) in the ratio 1:2 at AuNP concentration of 5.5 nM. The mixture was annealed at 40C for 10 minutes and cooled to room temperature. After the reaction, the composites were purified from excess ssDNA-QDs and ssDNA-AuNPs using gel electrophoresis.
Gel electrophoresis is a charge driven separation technique used for separating mixtures of charged components (e.g., ssDNAs (as they have negative phosphate backbone) from negatively charged NPs) based on their charge and/or size. In this technique, components to be separated are introduced in sample wells carved at the top of a gel that consists of small pores and pushed through it by applying an electric field across the gel. Each sample well forms a clear lane inside the gel permitting the separation of multiple samples simultaneously. According to the operating principle, smaller and/or highly charged components migrate faster through the pores compared to larger and/or less charged components, resulting in size and/or charge-based separation of the sample. Generally, the separation in each sample is dominated by either one of the charge or the size.
For analyzing the migration of a sample through the gel, individual components of the sample are spotted by imaging the gel after the electrophoresis run is complete. In our case, the QDs can be imaged for their fluorescence using a fluorescence scanner and the AuNPs can be imaged in a bright-field light setup, separately. In each of these images of the same gel, the corresponding NPs appear as dark bands in the bright background of sample wells (Fig 2).
In both the gel images (Fig 2 (A) and (B)), we introduced the QD-ssDNA conjugates and AuNP-ssDNA conjugates separately as individual samples in lane 1 and lane 3, respectively to act as the standards against which our actual unpurified QD-DNA-AuNP composite sample would be compared. The appearance of a dark band in the white box of Lane 1 (Fig2A) indicates the distance traveled by the QD-ssDNA conjugates alone whereas the presence of a dark band in the yellow box of Lane 3 (Fig2B) indicates the distance traveled by the AuNP-ssDNA conjugates alone in the gel. Therefore, we marked the yellow and white box across both the images as a reference for AuNP-ssDNA conjugates and QD-ssDNA conjugates that did not participate in the formation of QD-DNA-AuNP composites, respectively.
Our sample of interest, the unpurified QD-DNA-AuNP composite, was introduced in lane 2. Theoretically, the QD-DNA-AuNP composites should travel less in the gel as QD-DNA-AuNP composite is larger in size than the QD-ssDNA conjugates and the AuNP-ssDNA conjugates alone. Therefore, each lane 2 image (Fig 2 (A) and (B)) should consist of two bands: a band representing the excess NP-ssDNA conjugates at location comparable to the respective references and a farther up band representing the QD-DNA-AuNP composite. Although we do see the expected two bands in lane 2 of Fig 2 (A), lane 2 of Fig 2 (B) forms only one band parallel to the AuNP-ssDNA conjugates band in the reference lane. However, the QD and AuNP bands in the yellow box of lane 2 overlap which is only possible on the formation of QD-DNA-AuNP composite. Therefore, despite the discrepancy of the absence of two AuNP bands in lane 2, we think that QD-DNA-AuNP composite formation was successful. Currently, we hypothesize that we do not see two AuNP bands in lane 2 because the size contrary to our expectation did not solely dominate the separation in the gel. However, this current understanding of our system would be justified only after visually confirming the formation of QD-DNA-AuNP composite under a transmission electron microscope (TEM). We are currently in the process of synthesizing more QD-DNA-AuNP composites to image them in TEM. The results from this experiment would help us validate our current hypothesis and move towards the fluorescence quenching experiments in the future.