Photoswitching azobenzene modified DNA

February 20, 2019

by Abhilasha "Abby" Dehankar

Goal:  Our goal is to develop photoswitchable QD-DNA-AuNP composites that will enable us to use QDs for STORM imaging because of their predicted and 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.

The reversible photoswitching can be done multiple times depending on the effectiveness of azobenzene’s activity during each cycle. Further, control over the stochastic on/off behavior relies on the switchability of azobenzene-modified-deoxyribonucleic acid (azoDNA). Thus, azoDNA photoswitching investigation is imperative. This report summarizes our recent efforts to study the switchability of azoDNA without nanoparticles on current optical setup.

Figure 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 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. 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 our 2nd attempt at the synthesis of QD-DNA-AuNP conjugates using AuNP-ssDNA conjugates with high ssDNA labeling density formed by a new method. The success of composite formation was analyzed using gel electrophoresis, fluorescence, and optical imaging and indicated successful formation. However, visual confirmation with electron microscopy was yet to be undertaken.

Monthly Update:

This report includes an update on our recent efforts to study photoswitchability of azoDNA using the UV lamp in our fluorometer as the excitation source. For conducting this study, our first task was to determine a suitable detection system capable of capturing the hybridization-dehybridization of our azoDNA. DNA by nature displays a property called hyperchromicity, wherein single, dehybridized strands of DNA (ssDNA) absorbs more light in comparison to their hybridized form, double-stranded DNA (dsDNA). Changes in the absorbance over time can be readily recorded using a UV-visible spectrophotometer (UV-vis, available in-house). Further, hyperchromicity provides a perfect representation of the DNA switch by avoiding any interference from DNA modifiers required for other detection strategies, such as DNA modification with fluorophore-quencher pairs for FRET detection.

Next, we wanted to determine the capabilities of our detection system. For this, we decided to heat our azoDNA (a hybridized form of azo-modified DNA) above its melting point and test its response during hybridization while cooling over time with UV-vis. The absorbance was recorded at the peak absorption wavelength of DNA (260 nm). The underlying objectives for the experiment were: 1) To ensure that our azoDNA is functional, 2) To verify the abilities of our UV-vis to detect absorbance changes observed during hybridization-dehybridization of azoDNA, and 3) To determine the limits (absorbance in completely hybridized vs. completely dehybridized state) of our azoDNA.

Specifically, hybridized azoDNA was heated to temperatures (60-70 C) significantly above its melting point (42 C) at a 5 µM concentration and allowed to cool over 40 minutes to reach room temperature completing one heating cycle (Fig 2). Ideally, absorbance should be higher in dehybridized state after heating and decrease over time to the absorbance at room temperature in the hybridized state. The rate of absorption decrease also permits the study of azoDNA’s hybridization kinetics. We were able to successfully observe and detect the expected reduction in absorbance during all our repeated heating cycles confirming that our azoDNA is functional. Ideally, in a completely controlled system, the azoDNA absorption versus time curves for all heating cycles should overlay each other. Considering low control in our current method of experimentation, we did expect changes in the rate of decrease. However, our system should show the same absorbance after reaching room temperature. Unfortunately, we saw significant absorbance differences despite cooling to similar room temperatures. Additionally, the trend did not follow any particular order with respect to the highest and lowest temperatures of the system either making them unreliable for reproducibility of signals.

Figure 2: Absorbance verus time during the cooling of heated azoDNA from temperatures above it melting point to room temperature. Dehybridized DNA displays higher absorbance as compared to hybridized DNA

Despite this, since we did see desired differences in the absorbance of ssDNA and dsDNA during our heating cycles, we decided to try photoactivation of azoDNA with the fluorometer UV-lamp. The hybridized azoDNA was exposed to 365 nm for 70 minutes, and absorbance was measured at 10 minutes intervals. Exposure at 365 nm converts trans-azobenzene to cis-azobenzene, destabilizing and dehybridizing the DNA duplex into ssDNA that can be observed by an increase in absorbance. The experimental results (Fig 3) displayed erratic changes in absorbance as opposed to a continual rise even after prolonged exposure at 365 nm. This could be a result of insufficient energy supply by the employed UV lamp for promoting excitation or inefficiency of our detection system. We are still trying to narrow down the narrow down the exact reason. Next, our goal is to determine whether it is wiser to fix the problems in our current detection strategy or to move on to another method.

Figure 3: Absorbance verus time during photoactuation of hybridized azoDNA at 365 nm to drive trans-to-cis conversion of azobenzene and promote deybridization. Dehybridized DNA displays higher absorbance as compared to hybridized DNA