October 6, 2018
by Abhilasha "Abby" Dehankar
Goal: Our goal was to test the functionality of quantum dot (QDs) – single-stranded deoxyribonucleic acid (ssDNA) conjugates synthesized and described in our prior report. These QD-ssDNA conjugates should be able to bind to gold nanoparticles (AuNPs) labeled with complementary ssDNA through DNA hybridization to form QD-DNA-AuNP conjugates. The DNA employed in the resulting QD-DNA-AuNP conjugate contains photoresponsive azobenzene groups yielding the potential for reversible quenching of QD fluorescence using a mechanism described in Figure 1. These photoswitchable QD-DNA-AuNP conjugates will allow us to employ QDs for STORM imaging.
Review of progress-to-date:
Successful formation of QD-DNA-AuNP conjugates (Figure 1) requires nanoparticle (NP) conjugation to the ssDNAs and a configuration of conjugated ssDNAs that favor hybridization with its complementary pair on the adjacent NP. Prior to this update, we primarily tested the success and efficiency of ssDNA conjugation to QD-PC3 indirectly, by studying their fluorescence spectra. Briefly, we conjugated QD-PC3 with a fluorescent dye modified ssDNA. After purification from excess and unconjugated ssDNA, a successful conjugation of ssDNA to QD displayed fluorescence peaks from both the QD and the dye in the fluorescence spectra. However, this technique provided little information about the conformation of ssDNA in the conjugated state. Therefore, in this report, I discuss QD-ssDNA conjugation and configuration testing and associated results.
A popular method for detecting conjugation of ssDNA to NPs is through gel electrophoresis imaging. 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 charges and sizes. In this technique, components to be separated are pushed by an applied electric field through a gel consisting of small pores. Smaller components migrate faster through the pores compared to larger components, resulting in size-based separation.
Since conjugation to ssDNA results in an effective increase in the size of QDs (Figure 2), we tested the success of QD – ssDNA conjugation by comparing migration of conjugates to free QDs and ssDNA using gel electrophoresis. The migration of the QDs through the gel was detected by capturing their fluorescence. Fluorescent regions are indicated by dark regions/bands in the gel image. Figure 3 shows the fluorescence image of a gel consisting of 4 wells: QD-PC3 (1), ssDNA (2), empty (3) and unpurified QD – ssDNA conjugates (4), respectively, after separation. As expected, fluorescent bands of QDs were observed only in lanes (1) and (4). Since, smaller components move faster through the gel, they travel farther compared to larger components in the same electric field. Thus, migration of QD-PC3 (1) further down the well compared to the unpurified QD – ssDNA conjugates (4) indicated successful QD – ssDNA conjugation.
Testing the conformation
Next, we used the same gel electrophoresis imaging technique to test the conformation of conjugated ssDNA on QDs. Here, we introduced QD – ssDNA conjugates to a solution containing DNA origami hinges with complementary ssDNA strands present at a specific sites. If the ssDNA on the QD surface is in a desired configuration, hybridization with complementary strand on the hinge would be favored, leading to a substantial increase in the size of the final QD-DNA-DNA origami hinge (QD-hinge) conjugate compared to QD – ssDNA conjugates alone (Figure 4). To test this, the gel was prepared at high salt concentrations compared to the previous gel to maintain the stability of DNA origami hinges (which require salt). Migration was again observed by measuring the fluorescence of the QDs in the gel. However, as opposed to the previous gel image, the fluorescent regions in the gel were indicated by a bright region/band. This was because as opposed to previous images, these images were captured on an instrument with dark background setup.
Figure 5 shows the fluorescence image of a gel consisting of 3 wells: DNA origami hinges (1), QD – ssDNA conjugates (2) and DNA origami hinges + 2x QD – ssDNA conjugates (3), respectively. After separation, as expected, lane (1) that was devoid of QDs did not show any fluorescence except for a faint fluorescent band in the region of hinges. This fluorescence resulted from a minor auxiliary excitation of the fluorophores integrated in the hinges. Further, the QD – ssDNA conjugates (2) lane, displayed two distinct fluorescent bands. The extremely bright fluorescent band at the top of the lane near the well indicated aggregation of QD – ssDNA conjugates. Aggregation of QD – ssDNA conjugates results from decreased electrostatic repulsion between poorly passivated (low ssDNA per QD) QD – ssDNA conjugates at the high salt concentrations employed in this gel. In contrast, the bottom band displayed the highly stable QD – ssDNA conjugates that migrated through the gel without aggregation. As anticipated, we also observed similar bands in lane (3) from the aggregates QD-ssDNA conjugates and the stable QD – ssDNA conjugates that did not pair with the complementary strands of DNA origami hinges. However, apart from that, lane (3) was accompanied by formation of two distinct fluorescent bands. The lower band was observed at a region similar to that of the hinges in (1), but with significantly higher brightness. Additionally, this band migrated less in the gel compared to QD – ssDNA conjugates, as expected for successful conjugation to DNA origami hinges. Therefore, presence of this band indicated successful QD attachment to DNA origami hinges. The upper fluorescent band is generally observed because of aggregation of the QD-hinge composites. In conclusion, the ssDNA conjugated to the surface of QDs are available for hybridization with complementary pairs.
As a final test, we imaged the QD-hinge composites. Since the DNA origami hinges have complementary ssDNA sequences on the hinge arm only at a specific location, successful QD binding would be observed only at specific location on the arms on the hinges. We observed binding at this location, with occasional closing of the hinges by QDs (Figure 6A). Therefore, observation of QD-closed DNA origami hinges in TEM images clearly indicated successful formation of functional QD – ssDNA conjugates. Similarly, TEM images of AuNP-hinge composites (Figure 6B) indicated successful formation of a functional AuNP – ssDNA conjugates. Thus, with formation of all the functional components, future experiments will focus on synthesis and analysis of photoswitchable QD-DNA-AuNP conjugates.