Covalent Conjugation of Carbon Dots with Plasmid and DNA Condensation Thereafter: Realistic Insights into the Condensate Morphology, Energetics, and Photophysics

Abstract

The use of carbon quantum dots (CDs) as trackable nanocarriers for plasmid and gene as hybrid DNA condensates has gained momentum, as evident from the significant recent research efforts. However, the in-depth morphology of the condensates, the energetics of the condensation process, and the photophysical aspects of the CD are not well understood and often disregarded. Herein, for the first time, we covalently attached linearized pUC19 with citric acid and cysteamine-derived CD through the reaction of the surface amine groups of CDs with the 5′-phospho-methyl imidazolide derivative of the plasmid to obtain a 1:1 CD-pUC19 covalent conjugate. The CD-pUC19 conjugates were further transformed into DNA condensates with spermine that displayed a toroidal morphology with a diameter of ∼200 nm involving ∼2–5 CD-pUC19 conjugates in a single condensate. While the interaction of pristine CD to spermine was exothermic, the binding of the CD-pUC19 conjugate with spermine was endothermic and primarily entropy-driven. The condensed plasmid displayed severe conformational stress and deviation from the B-form due to the compact packing of the DNA but better transfection ability than the pristine CD. The CDs in the condensates tend to come close to each other at the core that results in their shielding from excitation. However, this does not prevent them from emanating reactive oxygen species on visible light exposure that compromises the decondensation process and cell viability at higher exposure times, calling for utmost caution in establishing them as nonviral transfecting agents universally.

Biomass derived self-assembled DNA-dot hydrogels for enhanced bacterial annihilation

“…The fluorescence quantum yield of DNA dots was found to be ∼4.1% with respect to quinine sulfate (QY = 0.55) in 0.5 M H 2 SO 4 , as a standard. 33 The DNA dots showed optimal excitation and emission wavelengths at 450 nm and 550 nm, respectively (Fig. 2D) with weak cyan-colored visible fluorescence on a transilluminator upon 365 nm irradiation.…”

“…To determine the quantum yield (QY) of the purified DNA dots comparatively, the following equation was employed: 33 Φ x = Φ s × ( I x f s η x 2 / I s f x η s 2 )where subscript x and s denote test and standard sample respectively; Φ is the fluorescence quantum yield; I is the integrated fluorescence intensity; f is the absorption factor ( f = 1–10-A, where A = absorbance); and η is the refractive index of the solvent (1.33 for water). Quinine sulfate (QY = 0.55) in 0.5 M H 2 SO 4 was used as a standard. f s = 0.06, I s = 1.23 × 10 8 , f x = 0.07, I x = 1.067 × 10 7 …”

“…Previous research has shown that many carbon dots (CDs) are adept at ROS generation on irradiation with normal light in the visible region. 22,23,33 Assuming the considerable pyrolization of the DNA into DNA dots similar to CDs, this prompted us to irradiate the DNA-dots in hydrogels with visible light from a tungsten source (100 Watts) to evaluate ROS generation using non-fluorescent DHR 123 as a probe that is converted to fluorescent rhodamine 123 on exposure to ROS (Fig. 4C).…”

“…Characterization of the DNA dot/hydrogel by gel electrophoresis and reverse phase HPLC 1% agarose gel electrophoresis was performed using TAE (Tris-Acetate-EDTA) buffer of pH 8, for 45 min at 90 volts and DNA bands were visualized after ethidium bromide staining. 22,33 However, DNA dots and PpIX control did not require any staining and could be visible with UV transillumination. Gel images were captured with a digital camera.…”

“…The efficiency of DNA dots, as well as the hydrogel for ROS generation, was studied in the presence of 5 µl, 2.5 nM dihydrorhodamine 123 (DHR123). 22,23,33 The samples were irradiated with a tungsten bulb (Philips, 100 W) at approx. 15 cm distance on the ice pack to maintain the temperature of the solution.…”