Plasmon-emitter coupling in cytosine-rich hairpin DNA-templated silver nanoclusters: Thermal reversibility, white light emission, and dynamics inside live cells.

Bio-templated luminescent noble metal nanoclusters (NCs) have attracted great attention for their intriguing physicochemical properties. Continuous efforts are being made to prepare NCs with high fluorescence quantum yield (QY), good biocompatibility, and tunable emission properties for their widespread practical applications as new-generation environment-friendly photoluminescent materials in materials chemistry and biological systems. Herein, we explored the unique photophysical properties of silver nanoclusters (AgNCs) templated by cytosine-rich customized hairpin DNA. Our results indicate that a 36-nucleotide containing hairpin DNA with 20 cytosine (C20) in the loop can encapsulate photostable red-emitting AgNCs with an absolute QY of ∼24%. The luminescent properties in these DNA-templated AgNCs were found to be linked to the coupling between the surface plasmon and the emitter. These AgNCs exhibited excellent thermal sensitivity and were employed to produce high-quality white light emission with an impressive color rendering index of 90 in the presence of dansyl chloride. In addition, the as-prepared luminescent AgNCs possessing excellent biocompatibility can effectively mark the nuclear region of HeLa cells and can be employed as a luminescent probe to monitor the cellular dynamics at a single molecular resolution.

Deciphering the liquid-liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein ß-lactoglobulin.

Owing to the significant role in the subcellular organization of biomolecules, physiology, and the realm of biomimetic materials, studies related to biomolecular condensates formed through liquid-liquid phase separation (LLPS) have emerged as a growing area of research. Despite valuable contributions of prior research, there is untapped potential in exploring the influence of phase separation on the conformational dynamics and enzymatic activities of native proteins. Herein, we investigate the LLPS of ß-lactoglobulin (ß-LG), a non-intrinsically disordered protein, under crowded conditions. In-depth characterization through spectroscopic and microscopic techniques revealed the formation of dynamic liquid-like droplets, distinct from protein aggregates, driven by hydrophobic interactions. Our analyses revealed that phase separation can alter structural flexibility and photophysical properties. Importantly, the phase-separated ß-LG exhibited efficient enzymatic activity as an esterase; a characteristic seemingly exclusive to ß-LG droplets. The droplets acted as robust catalytic crucibles, providing an ideal environment for efficient ester hydrolysis. Further investigation into the catalytic mechanism suggested the involvement of specific amino acid residues, rather than general acid or base catalysis. Also, the alteration in conformational distribution caused by phase separation unveils the latent functionality. Our study delineates the understanding of protein phase separation and insights into the diverse catalytic strategies employed by proteins. It opens exciting possibilities for designing functional artificial compartments based on phase-separated biomolecules.

Fluorescence Resonance Energy Transfer in a Supramolecular Assembly of Luminescent Silver Nanoclusters and a Cucurbit[8]uril-Based Host-Guest System.

The understanding of interactions between organic chromophores and biocompatible luminescent noble metal nanoclusters (NCs) leading to an energy transfer process that has applications in light-harvesting materials is still in its nascent stage. This work describes a photoluminescent supramolecular assembly, made in two stages, employing an energy transfer process between silver (Ag) NCs as the donor and a host-guest system as the acceptor that can find potential applications in diverse fields. Initially, we explored the host-guest chemistry between a cationic guest ethidium bromide and cucurbit[8]uril host to modulate the fluorescence property of the acceptor. The host-guest interactions were characterized by using UV-vis absorption, steady-state and time-resolved spectroscopy, molecular docking, proton 1H nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and isothermal calorimetry studies. Next, we prepared a series of blue-emitting AgNCs using different templates such as proteins and peptides. We have found that these AgNCs can be employed as a donor in the energy transfer process upon mixing with the above acceptor for emission color tuning. Our in-depth studies also revealed that surface ligands could play a key role in modulating the energy transfer efficiency. Overall, by employing a noncovalent strategy, we have tried to develop Förster resonance energy transfer (FRET) pairs using blue-emitting NCs and a host-guest complex that could find potential applications in constructing advanced sustainable light-harvesting, white light-emitting, and anti-counterfeiting materials.

DNA-Templated Modulation in the Photophysical Properties of a Fluorescent Molecular Rotor Auramine O by Varying the DNA Composition.

This work delineates an integrative approach combining spectroscopic and computational studies to decipher the association-induced fluorescence properties of a fluorescent molecular rotor, viz., auramine O (AuO), after interacting with 20-mer duplex DNA having diverse well-matched base pairs. While exploring the scarcely explored sequence-dependent interaction mechanism of AuO and DNA, we observed that DNA could act as a conducive scaffold to the formation of AuO dimer through noncovalent interactions at lower molecular density. The photophysical properties of AuO depend on the nucleotide compositions as described from sequence-dependent shifting in the emission and absorption maxima. Furthermore, we explored such DNA base pair-dependent fluorescence spectral characteristics of AuO toward discriminating the thermodynamically most stable single nucleotide mismatch in a 20-mer sequence. Our results are interesting and could be useful in developing analogues with further enhanced emission properties toward mismatched DNA sequences.

Preferential Binding of Epirubicin Hydrochloride with Single Nucleotide Mismatched DNA and Subsequent Sequestration by a Mixed Micelle.

Targeting mismatched base pairs containing DNA using small molecules and exploring the underlying mechanism involved during the binding interactions is one of the fundamental aspects of drug design. These molecules in turn are used in nucleic acid targeted therapeutics and cancer diagnosis. In this work, we systematically delineate the binding of the anticancer drug, epirubicin hydrochloride (EPR) with 20-mer duplex DNA, having both natural nucleobase pairing and thermodynamically least stable non-Watson-Crick base pairing. From the thermal denaturation studies, we observed that EPR can remarkably enhance the thermal stability of cytosine-cytosine (CC) and cytosine-thymine (CT) mismatched (MM) DNA over other 20-mer duplex DNA. From steady-state fluorescence spectroscopy and isothermal titration calorimetry studies, we concluded that EPR binds strongly with the mismatched duplex DNA through the intercalation binding mode. The interaction of EPR and duplex DNA has also been monitored at a single molecular resolution using fluorescence correlation spectroscopy (FCS). Dynamic quantitates such as diffusion coefficients and hydrodynamic radii obtained from an FCS study along with association and dissociation rate constants estimated from intensity time trace analyses further substantiate the stronger binding affinity of EPR to the thermally less stable mismatched DNA, formed by the most discriminating nucleobase (viz. cytosine). Additionally, we have shown that EPR can be sequestered from nucleic acids using a mixed micellar system of an anionic surfactant and a triblock copolymer. From thermal denaturation studies and circular dichroism spectroscopy, we found that the extent of drug sequestration depends on the binding affinity of EPR to the duplex DNA, and this mixed micellar system can be employed for the removal of excess drug in the case of a drug overdose.

Differentiating a Least-Stable Single Nucleotide Mismatch in DNA Via Metal Ion-Mediated Base Pairing and Using Thioflavin T as an Extrinsic Fluorophore.

Monitoring the DNA dynamics in solution has great potential to develop new nucleic acid-based sensors and devices. With spectroscopic approaches, both at the ensemble average and single-molecule resolution, this study is directed to differentiate a single nucleotide mismatch (SNM) via a metal ion-stabilized mismatched base-pairing (C-Ag+-C/C-Cu2+-T) (C = cytosine, T = thymine) and site-selective extrinsic fluorophore, specifically, Thioflavin T (ThT). This is the first approach of its kind where dynamic quantities like molecular diffusion coefficients and diffusion times have been utilized to distinguish the least-stable SNM (CC & CT) formed by the most discriminating nucleobase, specifically, cytosine in a 20-mer duplex DNA. Additionally, this work also quantifies metal ions (Ag+ and Cu2+) at lower concentrations using fluorescence correlation spectroscopy. Our results can provide greater molecular-level insights into the mismatch-dependent metal-DNA interactions and also illuminate ThT as a new fluorophore to monitor the dynamics involved in DNA-metal composites.

Pressure-induced assemblies and structures of graphitic-carbon sheet encapsulated Au nanoparticles.

A novel strategy of using hydrostatic pressures to synthesize gold-carbon (Au-C) nanohybrid materials is explored. The stable face-centered-cubic (fcc) Au undergoes a structural phase transition to a mixture of primitive orthorhombic and cubic phases as the carbon phase acquires a highly ordered onion-like carbon (OLC) structure which encapsulates the Au nanoparticles, thereby exerting an additional pressure. Increasing the pressure results in a one dimensional (1-D) chain-like structure with the primitive cubic Au nanoparticles contained in an amorphous carbon matrix. The OLC structure allows the formation of quenchable Au nanoparticle phases with the primitive close packing and Au-C hybrids with new mesoscopic structures. Under pressure, we observe the formation of a hybrid material composed of a poorly conducting matrix made of amorphous carbon and conducting OLC-encapsulated Au nanoparticles. The electrical conductivity of this hybrid material under pressure reveals a percolation threshold. We present a new synthesis approach to explore the interplay between atomic and mesoscopic structures and the electrical conductivity of metal hybrid structures.

Preferential Binding of Thioflavin T to AT-Rich DNA: White Light Emission through Intramolecular Förster Resonance Energy Transfer.

Herein we report the effect of different nucleobase pair compositions on the association-induced fluorescence enhancement property of Thioflavin T (ThT), upon binding with 20 base pair long double-stranded DNA (dsDNA). Analysis of binding and decay constants along with the association (Kass) and dissociation (Kdiss) rate constants obtained from the fluctuation in the fluorescence intensity of ThT after binding with different DNA revealed selective affinity of ThT toward AT-rich dsDNA. Molecular docking also substantiates the experimental results. We also observed that addition of orange-emitting ethidium bromide (EtBr) to cyan-emitting ThT-DNA complexes leads to bright white light emission (WLE) through Förster resonance energy transfer. Additionally, the emission of white light is far greater in the case of intra-DNA strands. Besides endorsing the binding insights of ThT to AT-rich dsDNA, the present investigations open a new perspective for realizing promising WLE from two biomarkers without labeling the DNA.

Egg-shell derived carbon dots for base pair selective DNA binding and recognition.

The development of base pair selective fluorescent binding probes and their interaction mode with nucleic acids have created great interest for sensing and biomedical applications. Herein, we have used chicken egg shell membrane (ESM) as a cost effective easily available protein source for the synthesis of highly fluorescent carbon dots. The detailed characterizations have confirmed the in situ formation of heteroatom doped graphitic carbon nanodots (CDs) from ESM. The intrinsic fluorescence property of the material has been utilized for the label free binding of duplex deoxyribonucleic acid (DNA). The interaction of different natural and synthetic DNAs with carbon dots resulted in the enhancement of fluorescence characteristics of the latter. Analysis of the binding data obtained from steady state fluorescence studies revealed a selective and stronger affinity of CDs to the adenine-thymine (AT) base pair rich double stranded DNA (ds DNA) than that of the guanine-cytosine (GC) pair rich ds DNA. Base pair specific binding was further validated from isothermal titration calorimetry (ITC) and melting temperature data. The thermodynamic profile revealed endothermic binding that was driven by the hydrophobic interaction at the nano-bio interfaces. The results reveal the potential of carbon dots as a new and promising fluorescent probe for base pair selective and sequence specific DNA recognition.

A new insight into the interaction of ZnO with calf thymus DNA through surface defects.

Experimental evidences on the binding interaction of ZnO and Calf Thymus (CT) DNA using several biophysical techniques are the centre of interest of the present study. The interaction of ZnO with CT DNA has been investigated in detail by absorption spectral study, fluorescence titration, Raman analysis, zeta potential measurement, viscometric experiment along with thermal melting study and microscopic analysis. Steady-state fluorescence study revealed the quenching (48%) of the surface defect related peak intensity of ZnO on interaction with DNA. The optimized concentration of ZnO and DNA to obtain this level of quenching has been found to be 0.049mM and 1.027µM, respectively. Additional fluorescence study with 8-hydroxy-5-quinoline (HQ) as a fluorescence probe for Zn2+ ruled out the dissolution effect of ZnO under the experimental conditions. DNA conjugation on the surface of ZnO was also supported by Raman study. The quantitative variation in conductivity as well as electrophoretic mobility indicated significant interaction of ZnO with the DNA molecule. Circular dichroism (CD) and viscometry titrations provided clear evidence in support of the conformational retention of the DNA on interaction with ZnO. The binding interaction was found to be predominantly entropy driven in nature. The bio-physical studies presented in this paper exploring ZnO-CT DNA interaction could add a new horizon to understand the interaction between metal oxide and DNA.

Selective Binding of Genomic Escherichia coli DNA with ZnO Leads to White Light Emission: A New Aspect of Nano-Bio Interaction and Interface.

Here, we report for the first time, a novel and intriguing application of deoxyribonucleic acid (DNA) in the area of optics by demonstrating white light emission by tuning the emission of a nanomaterial, ZnO rods, exhibiting surface defects, in the presence of genomic Escherichia coli DNA with a comparatively high quantum efficiency. In order to understand the DNA specificity, we have also studied the interaction of ZnO with CT, and ML DNA, ss EC DNA, synthetic polynucleotides and different mononucleosides and bases. Further, in order to understand the effect of particle shape and defects present in ZnO, we have also extended our study with ZnO rods prepared at higher temperature exhibiting red emission and ZnO particles exhibiting yellow emission. Interestingly, none of the above studies resulted in white light emission from ZnO-DNA complex. Our studies unequivocally confirmed that the concentration and the nature of DNA and ZnO together plays a crucial role in obtaining CIE coordinates (0.33, 0.33) close to white light. The much enhanced melting temperature (Tm) of EC DNA and the energetics factors confirm enhanced hydrogen bonding of ZnO with EC DNA leading to a new emission band. Our experimental observations not only confirm the selective binding of ZnO to EC DNA but also open a new perspective for developing energy saving light emitting materials through nano-bio interactions.

Unraveling the Interaction of Silver Nanoparticles with Mammalian and Bacterial DNA.

The focus of this study was to understand and unravel the interaction of silver nanoparticles (AgNPs) with different types of Deoxyribonucleic acid (DNA), mammalian and bacterial, having different base pair compositions. Binding of spherical silver nanoparticles (AgNPs) to Calf thymus (CT) DNA, Escherichia coli (EC) DNA and Micrococcus lysodeikticus (ML) DNA has been studied to gain insights into their mode of interaction and specificity. Interaction of AgNPs with synthetic DNA has also been carried out. On the basis of absorption, thermal melting, isothermal calorimetry and viscosity studies, we could establish the mode of binding and specificity of the synthesized silver nanoparticles with mammalian and bacterial DNA. Thermal melting (Tm) studies indicated a decrease in the Tm of all the DNAs, confirming the destabilization of DNA stacks on interaction with AgNPs. Comparative interaction studies with single stranded (ss) and double stranded (ds) DNAs further confirmed the specificity of the particles toward ds DNA. On the basis of the results we could confirm that the synthesized AgNPs could be used for selective detection of DNA through their DNA binding mechanism. In addition, the AgNPs-DNA complexes exhibited distinct differences in the SERS spectra making it an interesting SERS platform for identifying ds DNA. The optical and physical properties of AgNPs help in differentiating the DNAs of different base pair compositions through their binding affinity and specificity.

Selective detection of Escherichia coli DNA using fluorescent carbon spindles.

We investigate the interaction of hydrophilic blue emitting carbon spindles with various deoxyribonucleic acids (DNA) having different base pair compositions, such as Herring testes (HT), calf thymus (CT), Escherichia coli (EC) and Micrococcus lysodeikticus (ML) DNA, to understand the mode of interaction. Interestingly, the fluorescent carbon spindles selectively interacted with E. coli DNA resulting in enhanced fluorescence of the former. Interaction of the same carbon with other DNAs exhibited insignificant changes in fluorescence. In addition, in the presence of EC DNA, the D band in the Raman spectrum attributed to the defect state completely disappeared, resulting in enhanced crystallinity. Microscopy images confirmed the wrapping of DNA on the carbon spindles leading to the assembly of spindles in the form of flowers. Dissociation of double-stranded DNA occurred upon interaction with carbon spindles, resulting in selective E. coli DNA interaction. The carbon spindles also exhibited a similar fluorescence enhancement upon treating with E. coli bacteria. These results confirm the possibility of E. coli detection in water and other liquid foods using such fluorescent carbon.