Sagar Satpathi

A Green Solvent Induced DNA Package

Mechanistic details of DNA compaction is essential blue print for gene regulation in living organisms. Many in vitro studies have been implemented using several compaction agents. However, these compacting agents may have some kinds of cytotoxic effects to the cells. To minimize this aspect, several research works had been performed, but people have never focused green solvent, i.e. room temperature ionic liquid as DNA compaction agent. To the best of our knowledge, this is the first ever report where we have shown that guanidinium tris(pentafluoroethyl)trifluorophosphate (Gua-IL) acts as a DNA compacting agent. The compaction ability of Gua-IL has been verified by different spectroscopic techniques, like steady state emission, circular dichroism, dynamic light scattering and UV melting. Notably, we have extensively probed this compaction by Gua-IL through field emission scanning electron microscopy (FE-SEM) and fluorescence microscopy images. We also have discussed the plausible compaction mechanism process of DNA by Gua-IL. Our results suggest that Gua-IL forms a micellar kind of self aggregation above a certain concentration (≥1 mM), which instigates this compaction process. This study divulges the specific details of DNA compaction mechanism by a new class of compaction agent, which is highly biodegradable and eco friendly in nature.

Loading of an anti-cancer drug into mesoporous silica nano-channels and its subsequent release to DNA

Mesoporous silica nano-channel (MCM-41) based molecular switching of a biologically important anticancer drug, namely, ellipticine (EPT) has been utilized to probe its efficient loading onto MCM-41, and its subsequent release to intra-cellular biomolecules, like DNA. By exploiting various spectroscopic techniques (like, steady state fluorescence, time-resolved fluorescence and circular dichroism), it has been shown that EPT can be easily translocated from MCM-41 to DNA without using any external stimulant. Blue emission of EPT in a polar aprotic solvent, i.e., dichloromethane (DCM), completely switches to green upon loading inside MCM-41 due to the conversion from a neutral to a protonated form of the drug inside nano-pores. Powder X-ray diffraction (PXRD), N2 gas adsorption and confocal fluorescence microscopy results confirm the adsorption of EPT inside the nano-pores of MCM-41. Here, the lysozyme (Lyz) protein has been utilized as a pore blocker of MCM-41 in order to prevent premature drug release. Interestingly, EPT is released to DNA even from the EPT-MCM-Lyz composite system, and results in intensification of green fluorescence. Electron microscopy results reveal the formation of a distinctive garland kind of morphology involving MCM-41 and DNA probably through non-covalent interactions, and this is believed to be responsible for the DNA assisted release of drug molecules from silica nano-pores. Confocal laser scanning microscopy (CLSM) imaging revealed that EPT-MCM is successfully internalized into the HeLa cervical cancer cells and localized into the nucleus. Cell viability assay results infer that EPT-MCM and EPT-MCM-Lyz showed much improved efficacy in HeLa cancer cells compared to free ellipticine.

Ultrafast dynamics of a molecular rotor in chemical and biological nano-cavities

Molecular rotors have become indispensable tools in monitoring several important processes in chemistry and biology owing to their sensitivity towards viscosity. Despite their importance, less attention has been paid to understanding the excited state properties of molecular rotors. Recently, Maroncelli and coworkers unraveled the excited state photochemistry of a julolidine based molecular rotor, 9-(2-carboxy-2-cyano)vinyl julolidine (CCVJ), and claimed that CCVJ is not a simple rotor probe. Unlike other molecular rotors, photoisomerization is believed to be the main non-radiative decay pathway for this molecule. Inspired by their report, herein, we tried to understand how the excited state dynamics of CCVJ is affected inside the nano-cavities of cyclodextrins (CDs) and human serum albumin (HSA) protein using steady-state and femtosecond fluorescence up-conversion techniques. We observed a pronounced enhancement in fluorescence quantum yield when CCVJ is encapsulated in CDs (β- and γ-CD) and HSA. Femtosecond up-conversion studies reveal that the ultrafast dynamics of CCVJ are drastically retarded inside the nano-cavities of CDs and protein. All these results suggest that photoisomerization, which is believed to be the major non-radiative decay pathway of CCVJ, is severely restricted inside the abovementioned bio-mimetic and biological nano-cavities. The molecular images of orientations of CCVJ inside the nano-cavities of CDs and protein have been discussed by theoretical and molecular modeling studies. We believe the present results might be helpful in exploiting this molecule more in biological and viscosity sensing applications.

Spectroscopy and Dynamics of Cryptolepine in the Nanocavity of Cucurbit[7]uril and DNA

Herein, we explored the photophysical properties of the antimalarial, anticancer drug cryptolepine (CRYP) in the presence of the macrocyclic host cucurbit[7]uril (CB7) and DNA with the help of steady-state and time-resolved fluorescence techniques. Ground-state and excited-state calculations based on density functional theory were also performed to obtain insight into the shape, electron density distribution, and energetics of the molecular orbitals of CRYP. CRYP exists in two forms depending on the pH of the medium, namely, a cationic (charge transfer) form and a neutral form, which emit at λ=540 and 420 nm, respectively. In a buffer solution of pH 7, the drug exists in the cationic form, and upon encapsulation with CB7, it exhibits a huge enhancement in fluorescence intensity due to a decrement in nonradiative decay pathways of the emitting cryptolepine species. Furthermore, docking and quantum chemical calculations were employed to decipher the molecular orientation of the drug in the inclusion complex. Studies with natural DNA indicate that CRYP molecules intercalate into DNA, which leads to a huge quenching of the fluorescence of CRYP. Keeping this in mind, we studied the DNA-assisted release of CRYP molecules from the nanocavity of CB7. Strikingly, DNA alone could not remove the drug from the nanocavity of CB7. However, an external stimulus such as acetylcholine chloride was able to displace CRYP from the nanocavity, and subsequently, the displaced drug could bind to DNA.

Ionic liquid induced G-quadruplex formation and stabilization: spectroscopic and simulation studies

Among different polymorphs of DNA, G-quadruplex (GQ) formation in guanine rich sequences has received special attention due to its direct relevance to cellular aging and abnormal cell growths. To date, smaller ions like Na+, K+, Li+, and NH4+ are the best possible selective GQ stabilizing materials. Herein, we report that an ionic liquid (IL), i.e. guanidinium tris(pentafluoroethyl)trifluorophosphate, can not only instigate the GQ formation in the absence of conventional GQ forming ions (like Na+, K+, NH4+, etc.), but also stabilizes the GQ structure. This conformational transition has been confirmed through different spectroscopic tools and molecular dynamics (MD) simulation studies. MD simulation shows that one of the guanidinium cations resides in the G-tetrad core, while bulky anions prefer to stay near the GQ surface resulting in GQ formation and stabilization. This study thus brings out a special type of ionic liquid that acts as a GQ stabilizer. The origin of GQ stabilization by IL presented here may also help in the future design of IL for GQ formation and stabilization.

pH responsive translocation of an anticancer drug between cyclodextrin and DNA

Ellipticine, a well known anticancer drug, emits intense green color when it is intercalated in DNA. It exhibits blue color inside the nano-cavity of a supramolecular host, γ-cyclodextrin (γ-CD). Inspired by these unique fluorescence switching properties of the anticancer drug, in the present work we have monitored the interplay of the drug between γ-CD and DNA by varying the medium pH as a stimulus. Here, steady-state and picosecond time-resolved fluorescence as well as circular dichroism techniques are employed to decipher the location of the drug inside the γ-CD nano-cavity and DNA. Our results confirm that at higher pH the drug selectively stays at γ-CD, even in the presence of biopolymers and exhibits blue color; whereas at lower pH, it is preferentially located in DNA even in the presence of γ-CD and emits a green color. We believe this kind of pH driven translocation of drugs monitored by fluorescence switching may find possible applications in controlled release of the drug inside cells.