Analysis of DNA melting curves

June 19, 2019

by Abhilasha "Abby" Dehankar

Blog Editor: 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.

Figure 1. QD-DNA-AuNP conjugate reversible photoswitching mechanism. QD fluorescence quenches in the proximity of AuNPs through a mechanism called “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. 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.

Monthly Update:

The azobenzenes are incorporated by tethering them to additional sugar/phosphate linkages along the DNA backbone via a D-threoninol group. The incorporation of a trans-azobenzene stabilizes a DNA duplex by intercalation between the neighboring bases. This stabilization in the form of stacking interaction raises the melting temperature of the azobenzene-modified DNA above that of an otherwise identical native sequence. Actuation using UV light (320-370 nm) results in a transition of the azobenzene groups, promoting trans-to-cis photoisomerization. In the cis form, azobenzenes destabilize the DNA duplex, significantly lowering the melting temperature of the DNA. Blue light (~450 nm) converts the cis form back to trans, thereby permitting reversible optical control of DNA hybridization. However, successful switching is dependent on the quantum yield of azobenzene photoisomerization.

Photoinduced isomerization quantum yield of Azobenzene modified DNA is sensitive to the DNA sequence, melting point, number of azobenzene moieties on the DNA backbone as well as the local environmental conditions. It has been previously reported that incorporation of azobenzene in DNA significantly reduces the quantum yield of photoisomerization, thereby making the transitions from trans-cis and cis-trans difficult to accomplish. The azobenzene quantum yield is known to depend on the free volume surrounding the azobenzene site. This explains the decrease in quantum yield going from azobenzene free in solution to being incorporated in single-stranded DNA (ssDNA) to further decrease when attached in a double-stranded DNA (dsDNA).

 

 

Photoisomerization Quantum Yield

Azobenzene

100 units

Azobenzene in a single stranded DNA molecule

33.3 units

Azobenzene in a double stranded DNA molecule

2.2 units

Table 1. Photoisomerization quantum yield of Azobenzene moiety attached to different chemical structures

Due to low photoisomerization quantum yield observed for azobenzene modified dsDNA, it becomes necessary to have an idea of the melting point of our AzodsDNA in order to design operating parameters for a better photoswitching. For example, if we are operating at room temperature 200C, and cis-AzodsDNA has a melting point of 400C – In such case, exciting at 450 nm for cis to trans conversion wouldn’t be efficient considering the fact that the cis-duplex would not dissociate at room temperature. Hence, it is an aim of the experiment to determine the melting point of dsDNA both in trans as well as cis form.

The melting point of a dsDNA is defined as the temperature at which 50% of double-stranded DNA is changed to single-stranded DNA. The melting point of the DNA is computed by measuring the absorbance at 260 nm as a function of temperature. As the temperature is raised, the double strand begins to dehybridize into single strands leading to a rise in the absorbance intensity. Increased salt concentrations help diffuse negative repulsions between the phosphates in the DNA's backbone, thereby stability its helical structure. Thus, we observe an increase in the melting point of DNA in the presence of salt/buffer. The melting temperature is a function of DNA sequence, salt concentrations, and pH. The experiments were conducted at constant pH in PBS buffer or water.

Figure 2. Melting curves of AzodsDNA and Native dsDNA (without azobenzene) in PBS buffer

 

DNA

Melting Point (0C)

Native dsDNA in PBS buffer

56.53

Trans AzodsDNA in PBS buffer

63.05

Cis AzodsDNA in PBS buffer

38.02

Table 1. Melting temperatures for AzodsDNA and Native dsDNA in PBS buffer

Figure 3. Melting curves of AzodsDNA and Native dsDNA (without azobenzene) in Water

DNA

Melting Point (0C)

Trans AzodsDNA in Water

43.02

Cis AzodsDNA in Water

22.07

Table 2. Melting temperatures for AzodsDNA and Native dsDNA in Water

Conclusion: Melting curve analysis of DNA indicates that under the operating conditions followed for FRET studies, the melting point of Cis-AzodsDNA in PBS buffer is above room temperature (38.020C).

When trying to actuate using UV Light (450 nm) for cis to trans conversion, the majority of the DNA will be present in a double-stranded form. Due to low photoisomerization of Azobenzene in a double strand, it becomes highly unlikely that we will be able to switch from cis to trans conformation. The melting temperature being high won’t allow for complete dissociation of the double-stranded helical structure, thereby not allowing to photoswitch.

Future studies: For an efficient photoswitch, we require a DNA sequence which has a cis-melting point below room temperature and a trans-melting point well above room temperature. Previous studies1 have indicated that incorporating an equal number of azobenzene moieties on both sides of double-stranded DNA structure helps to achieve the goal.

 

References:

(1)     Liang, X.; Mochizuki, T.; Asanuma, H. A Supra-Photoswitch Involving Sandwiched DNA Base Pairs and Azobenzenes for Light-Driven Nanostructures and Nanodevices. Small 2009. https://doi.org/10.1002/smll.200900223.