Melting curve characterization of the new DNA sequence

August 14, 2019

by Abhilasha "Abby" Dehankar

Faiz's Update

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 report

Referring to the blog post of June, we characterized the melting point of a DNA having azobenzene attached to only one single strand of the double stranded duplex. We concluded that for an efficient photoswitch, it is required that the DNA should have a cis-melting point below room temperature and a trans-melting point well above room temperature. The previous DNA sequence did not satisfy the above criteria and hence, a new DNA sequence with azobenzene moieties attached to both the single strands was proposed.

The melting point of a dsDNA is defined as the temperature at which 50% of double-stranded DNA is converted to single-stranded DNA. The melting point of the DNA is computed by measuring the absorbance at 260 nm as a function of temperature. Single-stranded DNA absorbs more amount of light at 260 nm than double-stranded DNA. Hence, as the temperature is raised, the double strand begins to dehybridize into single strands leading to a rise in the absorbance intensity. The melting temperature is a function of DNA sequence, salt concentrations, and pH. The melting temperature experiments were conducted in PBS buffer (pH – 7.2, Na+ concentration - 100 mM).

Melting curve of double stranded DNA depicts a typical sigmoidal curve (Figure 2).

Figure 2. Typical melting curve of a DNA

The new DNA sequence procured is as follows:

1st strand:  CG  TXT   AXG   TXT   CA

2nd strand: GCXA  AXT   CXA  AX GT  (Complementary strand)

where X refers to the azobenzene moiety

Figure 3. Melting curve of the new DNA sequence (1 uM) analyzed using UV-Vis spectroscopy

DNA

Melting point (Tm)

Trans AzodsDNA

55.2 0C

Cis AzodsDNA

< 150C

Table 1. Melting point of Azo-dsDNA in trans- and cis- form

(AzodsDNA concentration – 1 uM, PBS buffer pH 7.2, 100 mM NaCl)

 

Melting temperature of dsDNA can be computed from the inflection point of the curve. Inflection point is defined as a point of a curve at which a change in the direction of curvature occurs. From figure 3., if we fit a sigmoidal curve to the plot, we observe that trans-AzodsDNA has a melting point of 55.20C. However, a similar sigmoidal curve fitting cannot be employed for cis-AzodsDNA. The cis-AzodsDNA does not depict a sigmoidal curve usually observed in melting experiments. From our data set, we can conclude that the melting point of cis-AzodsDNA is below 150C. However, if we were able to accurately analyze the absorbance curve for cis-AzodsDNA in a temperature range of below 00C to 150C, we would obtain a sigmoidal curve with an inflection point somewhere below 00C. Due to limitations of our operating instrument, it was not possible to accurately analyze the absorbance values at temperature range below 150C.

 

Using the same DNA sequence, Liang et. al. (2009)1 concluded that the melting temperature of Trans-AzodsDNA is 57.2 0C and Cis-AzodsDNA is below 00C. Hence, our results are similar to once reported in the paper. Such a DNA design sequence fits aptly to the melting requirements of DNA sequence required for an efficient photoswitching system.

 

Future studies: 1) Next set of experiments involve conjugating DNA strand to QD, with it complementary strand conjugated to gold nanoparticle.

2) Conducting FRET studies by incubating QD-ssDNA and Au-ssDNA

3) Demonstrating photoswitching of the above-mentioned system

 

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.