Abstract:Interaction studies between a set of poly(ethylene glycol) (PEG) based cationic bottle-brush block copolymers (BBCPs) and calf thymus DNA (ctDNA) were carried out using steady state fluorescence spectroscopy, UV melting experiments and dynamic light scattering measurements. Results suggested that these cationic block copolymers could effectively bind with negatively charged DNA. Although electrostatic interaction is believed to be the predominant contributing factor in the overall binding process, hydrophobic … Show more
“…6) and the effective diameters along with diffusion coefficients of the individual conjugates are provided in Table 1. The average hydrodynamic diameter of the flavone-DNA conjugate is found to be 145 nm which is smaller than literature values for free DNA (700 nm) [53]. This might be due to puckering in DNA upon binding with the ligand fisetin.…”
Our study determines the selectivity of graphene oxide (GO) to recognize its ligands (e.g. flavonoids) in facilitating the binding with their respective cellular targets. The polyhydroxy phenolic compounds, flavonoids, have a broad spectrum of therapeutic activities with high potency and low systemic toxicity. Despite the vast medicinal importance, their bioavailability is low. In this exploratory study, GO has been used as the transporter of three flavonols fisetin (3, 7, 3′, 4′-OH flavone), quercetin (3, 5, 7, 3′, 4′-OH flavone), and morin (3, 5, 7, 2′, 4′-OH flavone) for the physiological target DNA. Calf thymus DNA is chosen as the model physiological target. Characterization of GO is performed using FTIR, Raman and dynamic light scattering (DLS) spectroscopy. The strong absorption peak at 1730 cm−1 indicated the presence of carbonyl groups (C=O) at the edges of GO. The presence of sp3 carbons due to oxidation of sp2 carbons in GO is further proved by Raman spectroscopy. DLS provided the average size of the GO particles to be ~9 μm. The dual luminescence behavior of the flavonols has been used in this study for the noninvasive sensing of the GO-flavonol and GO-flavonol-DNA interactions; as well as for the selectivity of GO for one flavonol over other in transferring the ligand to DNA. Furthermore, circular dichroism (CD) indicated that the optical activity of GO undergoes drastic change when conjugated with flavonols. Molecular modeling corroborated the findings from the binding studies. GO provides high promise as facilitators for drug delivery.
“…6) and the effective diameters along with diffusion coefficients of the individual conjugates are provided in Table 1. The average hydrodynamic diameter of the flavone-DNA conjugate is found to be 145 nm which is smaller than literature values for free DNA (700 nm) [53]. This might be due to puckering in DNA upon binding with the ligand fisetin.…”
Our study determines the selectivity of graphene oxide (GO) to recognize its ligands (e.g. flavonoids) in facilitating the binding with their respective cellular targets. The polyhydroxy phenolic compounds, flavonoids, have a broad spectrum of therapeutic activities with high potency and low systemic toxicity. Despite the vast medicinal importance, their bioavailability is low. In this exploratory study, GO has been used as the transporter of three flavonols fisetin (3, 7, 3′, 4′-OH flavone), quercetin (3, 5, 7, 3′, 4′-OH flavone), and morin (3, 5, 7, 2′, 4′-OH flavone) for the physiological target DNA. Calf thymus DNA is chosen as the model physiological target. Characterization of GO is performed using FTIR, Raman and dynamic light scattering (DLS) spectroscopy. The strong absorption peak at 1730 cm−1 indicated the presence of carbonyl groups (C=O) at the edges of GO. The presence of sp3 carbons due to oxidation of sp2 carbons in GO is further proved by Raman spectroscopy. DLS provided the average size of the GO particles to be ~9 μm. The dual luminescence behavior of the flavonols has been used in this study for the noninvasive sensing of the GO-flavonol and GO-flavonol-DNA interactions; as well as for the selectivity of GO for one flavonol over other in transferring the ligand to DNA. Furthermore, circular dichroism (CD) indicated that the optical activity of GO undergoes drastic change when conjugated with flavonols. Molecular modeling corroborated the findings from the binding studies. GO provides high promise as facilitators for drug delivery.
“…On addition of protamine, a decrease in the intensity and red shift in the wavelength of CD spectra was observed. Such an alteration in the CD spectra corresponds to a conformational transition of B to ѱ-DNA, an essential condition for an ordered toroid-shaped DNA structure 12,32 . At higher protamine concentration, both the positive and negative bands were flattened confirming aggregation of DNA.Figure 2DNA Conformational studies.…”
DNA condensation is a ubiquitous phenomenon in biology, yet the physical basis for it has remained elusive. Here, we have explored the mechanism of DNA condensation through the protamine-DNA interaction, and by examining on it the influence of DNA binding drugs. We observed that the DNA condensation is accompanied by B to Ψ-DNA transition as a result of DNA base pair distortions due to protamine binding, bringing about the formation of toroidal structure through coil-globule transition. The binding energetics suggested that electrostatic energy, bending energy and hydration energy must play crucial roles in DNA condensation. EtBr intercalation interferes with the protamine-DNA interaction, challenging the distortion of the DNA helix and separation of DNA base pairs by protamine. Thus, EtBr, by competing directly with protamine, resists the phenomenon of DNA condensation. On the contrary, netropsin impedes the DNA condensation by an allosteric mechanism, by resisting the probable DNA major groove bending by protamine. In summary, we demonstrate that drugs with distinct binding modes use different mechanism to interfere with DNA condensation.
“…Similar observations were reported by us while studying the interaction of DNA with PEGylated cationic BCPs. 51 , 52 For a given charge ratio, the NIDM108 copolymer would have a higher concentration of temperature-sensitive PNIPA chains, helping in higher compaction of the polyplexes, which forced more EB to be released.…”
Successful intracellular delivery
of genes requires an efficient
carrier, as genes by themselves cannot diffuse across cell membranes.
Because of the toxicity and immunogenicity of viral vectors, nonviral
vectors are gaining tremendous interest in research. In this work,
we have investigated the temperature-dependent DNA condensation efficiency
of various compositions of a thermosensitive block copolymer viz.,
poly(N-isopropylacrylamide)-b-poly(2-(diethylamino)ethyl
methacrylate) (PNIPA-b-PDMAEMA). Three different
copolymer compositions of varying molecular weights were successfully
synthesized via the RAFT polymerization technique. Steady-state fluorescence
and circular dichroism (CD) spectroscopies, dynamic light scattering
(DLS) and zeta potential measurements, agarose gel electrophoresis,
and atomic force microscopy techniques were utilized to study the
interaction of the copolymers with DNA at temperatures above and below
the critical aggregation temperature (CAT). All these
experiments revealed that, above the CAT, there was
formation of highly stable and tight polymer–DNA complexes
(polyplexes). The size of polyplexes was dependent on the temperature
up to a certain charge ratio, as determined by the DLS results. The
results obtained from temperature-dependent fluorescence spectroscopy,
CD, and gel electrophoresis indicated that the DNA molecules were
shielded more from aqueous exposure above the CAT because of the formation of relatively more compact complexes. The
polyplexes also exhibited changes in the particle morphology below
and above the CAT, with particles generated above CAT being more spherical in morphology. These results suggested
at the possibility of modulating the complex formation by temperature
modification. The present biophysical studies would provide new physical
insight into the design of novel gene carriers.
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