DNA Condensation Triggered by the Synergistic Self-Assembly of Tetraphenylethylene-Viologen Aggregates and CT-DNA

Development of small organic chromophores as DNA condensing agents, which explore supramolecular interactions and absorbance or fluorescence-based tracking of condensation and gene delivery processes, is in the initial stages. Herein, we report the synthesis and electrostatic/groove binding interaction–directed synergistic self-assembly of the aggregates of two viologen-functionalized tetraphenylethylene (TPE-V) molecules with CT-DNA and subsequent concentration-dependent DNA condensation process. TPE-V molecules differ in their chemical structure according to the number of viologen units. Photophysical and morphological studies have revealed the interaction of the aggregates of TPE-V in Tris buffer with CT-DNA, which transforms the fibrous network structure of CT-DNA to partially condensed beads-on-a-string-like arrangement with TPE-V aggregates as beads via electrostatic and groove binding interactions. Upon further increasing the concentration of TPE-V, the “beads-on-a-string”-type assembly of TPE-V/CT-DNA complex changes to completely condensed compact structures with 40–50 nm in diameter through the effective charge neutralization process. Enhancement in the melting temperature of CT-DNA, quenching of the fluorescence emission of ethidium bromide/CT-DNA complex, and the formation of induced CD signal in the presence of TPE-V molecules support the observed morphological changes and thereby verify the DNA condensation abilities of TPE-V molecules. Decrease in the hydrodynamic size, increase in the zeta potential value with the addition of TPE-V molecules to CT-DNA, failure of TPE-V/cucurbit(8)uril complex to condense CT-DNA, and the enhanced DNA condensation ability of TPE-V2 with two viologen units compared to TPE-V1 with a single viologen unit confirm the importance of positively charged viologen units in the DNA condensation process. Initial cytotoxicity analysis on A549 cancer and WI-38 normal cells revealed that these DNA condensing agents are non-toxic in nature and hence could be utilized in further cellular delivery studies.

measurements in each run and the mean value is used to construct the plot of size, zeta-potential versus concentration of TPE-V.
CT-DNA samples were prepared by dissolving the commercially available (Sigma Aldrich) white fibrous mass in de-ionized Milli Q water. Initially, the solution was vortexed for 2-3 minutes and allowed to stand overnight at 0-4 o C to completely dissolve the fibrous structure. Later, the samples were filtered through 0.45 mm syringe filters and concentrations were calculated using the average ε value (6600 M -1 cm -1 ) of single nucleotide at 260 nm. All the DNA-binding studies described in this work are carried out in 10 mM Tris buffer containing 2mM NaCl. 1 mM stock solutions of TPE-V were prepared by adding Milli Q water to appropriately weighed samples and heated to dissolve. From the hot stock solutions, concentrations required for studies were syringed out into cuvette and diluted to 1mL with Tris buffer. These solutions of TPE-V were incubated at room temperature for 30-40 minutes before performing DNA binding studies. This incubation time were chosen by monitoring the absorption changes to get a constant absorption for the aggregate band of TPE-V.

AFM/TEM sample preparations
Samples for AFM analysis were prepared by drop-casting 5-7 L solution over freshly cleaved mica surface, followed by slow air drying. The images were recorded at ambient conditions using a BRUKER MULTIMODE AFM operating in the tapping mode. NT-MDT-NSG series, TiN cantilever tips with 299 kHz resonance frequency was used for the analysis. TEM samples were prepared by dropcasing 20-30 L samples over carbon coated copper grid (400 meshes, Ted Pella) and air dried for 2 days at ambient conditions followed by vacuum drying before imaging. TEM images were recorded with JEOL-JEM0310 microscope with an accelerating voltage of 100 kV and the samples were imaged using a Hamamatsu ORCA charge-coupled device (CCD) camera.

Cell culture and MTT assay
Cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM, Himedia) added with 10% fetal bovine serum (FBS) and 1% Antibiotics Antimycotic solution (1X) at 37 °C with 5% CO2. The cells were trypsinised and seeded in a 96 well flat bottom microlitre plate at a density of 2.2. Synthesis of (2-(p-tolyl)ethene-1,1,2-triyl)tribenzene, (2) (Ma et al., 2018) Into a 250 ml three necked RB flask, 4-methylbenzophenone (3.0116 g, 15.2 mmol), benzophenone (2.8191 g, 15.2 mmol) and Zn dust (8.906 g, 136 mmol) were added. The flask was evacuated and flushed with argon 3 times. After the addition of dry THF (150 mL), reaction mixture was cooled in ice bath to 0 o C and TiCl4 (7.5 mL, 68.4 mmol) was slowly injected. The mixture was stirred at room temperature for 30 minutes and then refluxed under argon condition for 48 hrs. The mixture was then extracted with ethyl acetate and organic part was dried over anhydrous Na2SO4. The crude product was purified by silica column chromatography using hexane as eluent to give a white solid. Yield = 73% (3.8796 g).

Synthesis of (2-(4-(bromomethyl)phenyl)ethene-1,1,2-triyl)tribenzene, (3)
1 (1.5825 g, 4.4 mmol), freshly recrystallized NBS (0.9936 g, 5.1 mmol) and catalytic amount of BPO (0.015 g) were taken in a 100 mL RB flask and 60 mL of CCL4 was added. The solution was refluxed for 24 hours before it was cooled to room temperature. The precipitate was filtered and washed with dichloromethane. The filtrate was extracted with dichloromethane, and the organic fraction was dried under reduced pressure. The crude product thus obtained was further purified with silica gel (230-400 mesh) column chromatography using 2% ethyl acetate-hexane as eluent to yield a white solid.