Azobenzene and the actuation system

November 6, 2018

by Abhilasha "Abby" Dehankar

Goal: Our goal was to study the switchability of the photoresponsive, hybridized azobenzene-modified-deoxyribonucleic acid (azoDNA) without the nanoparticles on our current optical setup. AzoDNA consists of two complementary pairs of single stranded DNA (ssDNA), one of which is modified with azobenzene molecules. The azo-modified-ssDNA would be used to label the gold nanoparticles (AuNPs) and the complementary unmodified-ssDNA quantum dots (QDs). This should allow the formation of QD-azoDNA-AuNP conjugates on hybridization of the azoDNA. The azoDNA should allow 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.

CaptionFigure 1: QD-DNA-AuNP conjugate reversible photoswitching mechanism.
QD fluorescence quenches in proximity of AuNPs through a mechanism called as
“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 the organic phase QDs purchased from NN-labs to the aqueous phase. We achieved the transfer by exchanging the non-polar ligands on the QD surface with polar phytochelatin-3 (PC3). The resulting QDs (QD-PC3) are water soluble and terminated with carboxylate (-COOH) and amine (-NH2) groups on their surfaces

2) We successfully conjugated dibenzocyclooctyne (DBCO) to -NH2 terminations on the surface of QD-PC3 to form QD-DBCO conjugates using commonly-employed “carbodiimide” chemistry. Carbodiimide chemistry forms an amide (-CONH2-) bond between the –COOH groups and -NH2 groups. This allowed us to employ “click” chemistry to successfully conjugate ssDNA to QD-PC3. To implement this approach, we mixed the QD-DBCO conjugates with azide (-N3) terminated ssDNA to permit strain promoted alkyne - azide cycloaddition via a click reaction, generating QD-ssDNA conjugates.

3) In the previous report, we used the DNA origami platform to test the functionality of the QD-ssDNA and AuNP-ssDNA conjugates. For this, we mixed the nanoparticle (NP) – ssDNA conjugates with DNA origami hinges with corresponding complementary ssDNA sequences only at a specific location on the hinge arm. Successful NP binding would be observed only at specific location on the arms on the hinges indicating functional NP-ssDNA conjugates.

Monthly Update:

During the previous report cycle, we tested the functionality of both the NP-ssDNA conjugates and observed that they could bind to the corresponding complementary pair. Therefore, our next goal was to form the QD-azoDNA-AuNP conjugates. For achieving this goal, it is extremely important to precisely determine the concentrations of each of the individual components. As mentioned in one of our prior report (Does DNA sequence matter during QD- DNA conjugation?), in our current protocol we purify the QD-ssDNA conjugates from excess, unconjugated ssDNA size based separation method. Although successful in purification, this method is causing a significant loss and dilution of quantum dots during the process (~90-95%). Such a low yield of QD-ssDNA makes it challenging to accurately determine the concentration for our experiments. In addition to synthesis of the QD-azoDNA-AuNP conjugates, our goal for the project involves development of an optimized process to achieve these conjugates. Therefore, currently I am investigating alternative purification techniques (eg: Gel separation, Centrifugal filtration, etc.) that results in higher yield in addition to successful purification.

Meanwhile, I decided to perform a preliminary test on our optical setup using just the photoswitchable hybridized azoDNA without any NPs. Investigating the capabilities of our current setup for the actuation and detection of the photoswitchable azoDNA is important before we proceed with the studies on our QD-azoDNA-AuNP conjugates for two reasons:

1) Determination of required changes in the setup.

2) Studying the photoswitching abilities of the azoDNA.

To enable this study, I used a azoDNA with a fluorophore molecule (Cyanine 5, Cy5) attached to one end of the unmodified ssDNA and a quencher molecule (Q) attached to the corresponding complementary azobenzene-modified-ssDNA to form a fluorophore-quencher pair that is separated by the length of the DNA (Cy5-azoDNA-Q) as expected for our QD-azoDNA-AuNP conjugates (Fig 2A). The fluorophore (Cy5)-quencher (Q) pair in close proximity leads to a decrease in the fluorescence of the fluorophore through a mechanism called Forster Resonance Energy Transfer (FRET) (similar to the description in caption of Fig 1). For optimal FRET, the fluorescence spectra of the fluorophore should overlap completely with the absorbance spectra of the quencher. Therefore, for this study, we used a quencher with an absorption spectra that completely overlaps the emission spectra of Cy5 (Fig 2B).

Figure 2: (A) Cy5-azoDNA-Q pair photoswitch mechanism, (B) Absorbance of Q and fluorescence of Cy5 showing maximum overlap

Next, the Cy5-azoDNA-Q pair was prepared for the actuation studies by mixing the Cy5-ssDNA with complementary Q-azossDNA at an equivalent molar ratio (1:1) at a concentration of 125 nM each. The Cy5-ssDNA fluorescence signal quenching was measured by recording its fluorescence spectra (Excitation: 649 nm, Emission: 654-700 nm) before and after addition of the complementary Q-azossDNA at the same concentration (125 nM, Fig 3). We observed a significant quenching in the peak fluorescence intensity (~80%) on addition of the quencher.

Figure 3: Quenching of fluorescence intensity if Cy5-ssDNA on formation of Cy5-azoDNA-Q pair

To begin with I had a hybridized Cy5-azoDNA-Q pair as it was prepared in natural white/visible light (400-700 nm) making the fluorescence peak intensity at the beginning our “off” state. For this report, I focused only on the transition from “off” state to “on” state. As stated earlier, this can be achieved by actuation in the UV-range (200-400 nm) that dehybridizes the Cy5-azoDNA-Q pair by isomerization of the incorporated azobenzene to a cis form that destabilizes the hybridized state. I used an external UV-lamp (actuation wavelength- 365 nm) as the actuation source with 5 minutes as the exposure time per interval for this experiment. The dehybrization was detected by recording the fluorescence spectra of Cy5 (Excitation: 649 nm, Emission: 654-700 nm). We expect an increase in the peak fluorescence of Cy5 on UV actuation. We observed negligible changes in the peak fluorescence of Cy5 on UV exposure for a consecutive 10 minutes exposure. This raises of the question of whether the actuation source is powerful enough for azobenzene isomerization. Although, we had already modified our fluorometer with an external laser with the help of our Optics team at Georgia at the beginning of this term, the plan was to use the laser as the excitation source. Therefore, the real question now is whether we need to invest in a new UV-laser to act as a more powerful actuation source.