NIR-emissive carbon nanodots as a tool to mark ribosomal RNA and nucleolus components using super-resolution microscopy.

Ribosomal RNA (rRNA) plays a key role in protein synthesis and ribosomal biogenesis. The exclusively used commercial dye for RNA staining is SYTO RNASelect, which works in fixed cells only. To overcome this constraint, we synthesized NIR-emissive, highly photostable, and biocompatible carbon nanodots (CNDs) as a fluorescent biomarker for rRNA. The synthesized CNDs could stain rRNA in both live and fixed cells. We were able to visualize rRNA at different sites in eukaryotic cells using super-resolution microscopy (SRM). The CNDs localized rRNA in the dense fibrillar components (DFCs) of the nucleolus, nuclear membrane, and rough endoplasmic reticulum (RER). The super-resolved hollow ring-structured DFC with an FWHM of 140 nm, nuclear membrane with an FWHM of 120 nm, and ER with an FWHM of 115 nm were observed. We further found a marked contrast between the pre-RNA synthesized in cancer cells and normal cells. We believe that these CNDs have great potential in rRNA imaging and comprehending the complex relationships between rRNA dynamics and basic biological processes, disease development, or drug interactions.

Super-Resolution Microscopy Unveils Synergistic Structural Changes of Organelles Upon Point Mutation.

Ethyl methanesulphonate (EMS), is a widely used chemical mutagen that causes high-frequency germline null mutation by inserting an alkyl group into the nucleotide guanine in eukaryotic cells. The effect of EMS on the dynamics of the aneuploid genome, increased cellular instability, and carcinogenicity in relation to benign and malignant tumors are reported, but the molecular level understanding of morphological changes of higher-order chromatin structure has poorly been understood. This is due to a lack of sufficient resolution in conventional microscopic techniques to see small structures below the diffraction limit. Here, using super-resolution radial fluctuation, a largely fragmented, decompaction, and less dense heterochromatin structure upon EMS treatment to HEK 293A cells without any change in nuclear DNA domains is observed. This result suggests an early stage of carcinogenicity happened due to the point mutation. In addition, the distinct structural changes with an elongated morphology of lysosomes are also observed. On the other hand, fragmented and increased heterogeneous populations with an increased cytoplasmic occupancy of mitochondria are observed.

Tracking the super resolved structure of mitochondria using red emissive carbon nanodots as a fluorescent biomarker.

Herein, we report new red emissive highly photostable and water-soluble carbon nanodots (TPP CNDs) to visualize mitochondrial dynamics using super-resolution radial fluctuations (SRRF) microscopy. The TPP CNDs were synthesized in a one-step method, using 3-(carboxypropyl)triphenylphosphonium bromide (TPP) and o-phenylenediamine (OPDA) as precursors. The obtained crystal structure, NMR, and mass data suggested the presence of [3-(1H-benzimidazol-2-yl)propyl](triphenyl)phosphonium bromide (C28H26N2P+Br-) as a molecular fluorophore (MF) on the surface of the TPP CNDs. The TPP CNDs showed better photostability than the commercially available MitoTracker™ Green and were highly capable for long-term imaging of mitochondrial fission during hyperglycemic conditions and structural changes upon an antidiabetic drug treatment, without altering their fluorescence nature.

Unveiling the Long-Lived Emission of Copper Nanoclusters Embedded in a Protein Scaffold.

Protein-conjugated coinage metal nanoclusters have become promising materials for optoelectronics and biomedical applications. However, the origin of the photoluminescence, especially the long-lived excited state emission in these metal nanoclusters, is still elusive. Here, we unveiled the underlying mechanism of long-lived emission in albumin protein-conjugated copper nanoclusters (Cu NCs) using steady state and time-resolved spectroscopic techniques. Our findings reveal room-temperature phosphorescence (RTP) in protein-conjugated Cu NCs. Time-resolved area-normalized spectra distinguished short- and long-lived components, where the former arises from the singlet state and the latter from the triplet state, thus resulting in RTP. The similarity of the emission spectra at room (298 K) and cryogenic (77 K) temperature ascertains the RTP phenomenon by harvesting the higher-lying triplet states. Time-gated bioimaging of A549 cells using the long-lived emission not only supports RTP emission in the cellular environment but also provides exciting avenues in long-term bioimaging using bovine serum albumin-conjugated Cu NCs.

Mapping the time dependent DNA fragmentation caused by doxorubicin loaded on PEGylated carbogenic nanodots using fluorescence lifetime imaging and superresolution microscopy.

Doxorubicin is an anthracycline drug most commonly used in cancer therapy. It intercalates with the nuclear DNA and induces toxicity by causing DNA breaks and histone eviction. However, the kinetics of its action on the nucleus has not been mapped effectively. This study shows successful PEGylation and DOX loading through π-π interaction onto carbogenic fluorescent nanodots (FNDs), which have an affinity for the nucleolus. Then the drug release from the nanoparticle and its action on the nuclear environment were aptly mapped using both fluorescence lifetime imaging and superresolution radial fluctuation (SRRF) techniques. Here for the first time, the nuclear degradation kinetics caused by the released DOX from the FNDs as a result of DNA double-strand breaks and histone eviction was visualized. This led to the observation of decreasing length, breadth, and complex structure of the nuclear clusters from 6 h to 24 h, resulting in isolated cluster visualization. However, the superresolution images for free DOX and untreated cells reveal no such drastic effects at the same concentration and time points, unlike DOX loaded particles.

SARS-CoV-2 Spike mutations modify the interaction between virus Spike and human ACE2 receptors.

The high mutability of the SARS-CoV-2 virus is a growing concern among scientific communities and health professionals since it brings the effectiveness of repurposed drugs and vaccines for COVID-19 into question. Although the mutational investigation of the Spike protein of the SARS-CoV-2 virus has been confirmed by many different researchers, there is no thorough investigation carried out at the interacting region to reveal the mutational status and its associated severity. All the energetically favorable mutations and their detailed analytical features that could impact the infection severity of the SARS-CoV-2 virus need to be identified. Therefore, we have thoroughly investigated the most important site of the SARS-CoV-2 virus, which is the interface region (Residue 417-505) of the virus Spike that interacts with the human ACE2 receptor. Further, we have utilized molecular dynamic simulation to observe the relative stability of the Spike protein with partner ACE2, as a consequence of these mutations. In our study, we have identified 52 energetically favorable Spike mutations at the interface while binding to ACE2, of which only 36 significantly enhance the stabilization of the Spike-ACE2 complex. The stability order and molecular interactions of these mutations were also identified. The highest stabilizing mutation V503D confirmed in our study is also known for neutralization resistance.

Structural and spectroscopic characterization of pyrene derived carbon nano dots: a single-particle level analysis.

The bottom-up approach has been widely used for large-scale synthesis of carbon nanodots (CNDs). However, the structure and origin of photoluminescence in CNDs synthesized by the bottom-up approach is still a subject of debate. Here, using a series of separation techniques like solvent extraction, column chromatography, gel electrophoresis and dialysis, we present three distinct fluorescent components in CNDs synthesized from pyrene, a well-known precursor molecule. The separated components have qualitative and quantitatively different absorption and emission spectral features including quantum yield (QY). Optical and vibrational spectroscopy techniques combined with electron microscopy indicate that a subtle balance between the extent of graphitization and the presence of molecular fluorophores determines the nature of fluorescence emission. A substantial difference in photons/cycle, single-particle fluorescence blinking, ON-OFF photoswitching strongly supports the distinct nature of the components.

Development of a Highly Specific, Selective, and Sensitive Fluorescent Probe for Detection of Mycobacteria in Human Tissues.

Tuberculosis (TB), including extrapulmonary TB, is responsible for more than one million deaths in a year worldwide. Existing methods of mycobacteria detection have poor sensitivity, selectivity, and specificity, especially in human tissues. Herein, the synthesis of a cholic acid-derived fluorescent probe (P4) that can specifically stain the mycobacterium species is presented. It is shown that P4 probe specifically binds with mycobacterial lipids, trehalose monomycolate, and phosphatidylinositol mannoside 6. P4 probe can detect mycobacteria in polymicrobial planktonic cultures and biofilms with high specificity, selectivity, and sensitivity. Moreover, it can detect a single mycobacterium in the presence of 10 000 other bacilli. Unlike the probes that depend on active mycobacterial enzymes, the membrane-specific P4 probe can detect mycobacteria even in formalin-fixed paraffin-embedded mice and human tissue sections. Therefore, the ability of the P4 probe to detect mycobacteria in different biological milieu makes it a potential candidate for diagnostic and prognostic applications in clinical settings.

Structural Decoding of a Small Molecular Inhibitor on the Binding of SARS-CoV-2 to the ACE 2 Receptor.

Inhibition of the interaction of the receptor-binding domain (RBD) of the spike protein and the human angiotensin-converting enzyme 2 (ACE 2) receptor is the most effective therapeutic formulation to restrict the contagious respiratory illness and multiple organ failure caused by the novel SARS-CoV-2 virus. Based on the structural decoding of the RBD of the spike protein, here we have generated a new set of small molecules that have strong inhibiting properties on the binding of the spike protein to ACE 2 receptors. These small-molecule inhibitors surprisingly show binding to the main protease, nucleoprotein, and RNA-dependent RNA polymerase, which are the other responsible factors for the viral infection. The newly designed molecules show better performance than several existing repurposed drugs. Conformational changes from closed to closed lock and open conformations of the SARS-CoV-2 binding to the ACE 2 receptor were observed in the presence of these small molecular inhibitors, suggesting their strong abilities to counteract the SARS-CoV-2 infection.

Direct visualization of the protein corona using carbon nanodots as a specific contrasting agent.

The cellular uptake of the nanoparticles is greatly affected by the formation of protein corona. As a result, an in-depth knowledge of direct visualization of the corona and quantification thereof is extremely important. Although transmission electron microscopy is one of the best techniques for visualization, the heavy metals that are used to increase the contrast of protein are non-specific and may lead to artifacts and erroneous conclusions. Here, we present a new strategy using carbogenic nanodots that showed excellent contrast, under a transmission electron microscope for the direct visualization and quantification of the single particle protein corona.

Magnetofluorescent Nanoprobe for Multimodal and Multicolor Bioimaging.

Although, superparamagnetic iron oxide nanoparticles (SPIONs) have extensively been used as a contrasting agent for magnetic resonance imaging (MRI), the lack of intrinsic fluorescence restricted their application as a multimodal probe, especially in combination with light microscopy. In Addition, the bigger size of the particle renders them incompetent for bioimaging of small organelles. Herein, we report, not only the synthesis of ultrasmall carbon containing magneto-fluorescent SPIONs with size ∼5 nm, but also demonstrate its capability as a multicolor imaging probe. Using MCF-7 and HeLa cell lines, we show that the SPIONs can provide high contrast mulicolor images of the cytoplasm from blue to red region. Further, single particle level photon count data revealed that the SPIONs could efficaciously be utilized in localization based super resolution microscopy in future.

Intrinsically disordered proteins of viruses: Involvement in the mechanism of cell regulation and pathogenesis.

Intrinsically disordered proteins (IDPs) possess the property of inherent flexibility and can be distinguished from other proteins in terms of lack of any fixed structure. Such dynamic behavior of IDPs earned the name "Dancing Proteins." The exploration of these dancing proteins in viruses has just started and crucial details such as correlation of rapid evolution, high rate of mutation and accumulation of disordered contents in viral proteome at least understood partially. In order to gain a complete understanding of this correlation, there is a need to decipher the complexity of viral mediated cell hijacking and pathogenesis in the host organism. Further there is necessity to identify the specific patterns within viral and host IDPs such as aggregation; Molecular recognition features (MoRFs) and their association to virulence, host range and rate of evolution of viruses in order to tackle the viral-mediated diseases. The current book chapter summarizes the aforementioned details and suggests the novel opportunities for further research of IDPs senses in viruses.

Labelling Proteins with Carbon Nanodots.

We present efficient labelling of several proteins with orange-emissive carbon dots. N-Hydroxysuccinimide was used to activate the carboxyl groups of carbon dots, which subsequently reacted with the lysine groups present on the protein. Labelling was confirmed by UV absorption spectroscopy, PAGE and fluorescence correlation spectroscopy. Protein-conjugated carbon dots showed an enhancement in fluorescence lifetime and intensity owing to reduced intramolecular dynamic fluctuations. Single-molecule fluorescence measurements showed reduced fluorescence fluctuations and higher photon budget after protein tagging. Our study opens up opportunities to use carbon dots as highly precise biolabelling probes.

Carbon dots for naked eye colorimetric ultrasensitive arsenic and glutathione detection.

A novel one-step method for the synthesis of bright, multicolor fluorescent sulphur doped carbon dots (CNDs) has been developed by using simple microwave assisted pyrolysis of citric acid and sodium thiosulphate. The synthesized CNDs showed dual mode naked eye colorimetric ultrasensitive sensing capability both for arsenic [As (III)] and glutathione (GSH) with high selectivity. Using fluorometric assay, the detection limit (DL) for As (III) was found to be as low as 32pM. The selectivity data show that the newly developed CNDs is very specific for As (III) even with interference by high concentrations of other metal ions. The CNDs were also able to detect GSH very selectively over other biothiols like cysteine (Cys) and homo-cysteine (H-cys) with a DL of 43nM, even in blood plasma. The fast kinetic data suggests that the present CNDs assay could be used onsite As (III) detection. The CNDs, further, showed its potential application in high resolution bioimaging of bacterial nucleoid segregation.

Nitrogen-doped, thiol-functionalized carbon dots for ultrasensitive Hg(II) detection.

Nitrogen-doped, PEGylated carbon dots (C-dots) have been synthesized for the detection of mercury ions (Hg(2+)). The detection limit was found to be 6.8 nM. However, upon functionalization with dithiothreitol (DTT), it reached to as low as 18 pM. The C-dots-Hg(2+) system was also able to efficiently detect biothiols.

Reversible Photoswitching of Carbon Dots.

We present a method of reversible photoswitching in carbon nanodots with red emission. A mechanism of electron transfer is proposed. The cationic dark state, formed by the exposure of red light, is revived back to the bright state with the very short exposure of blue light. Additionally, the natural on-off state of carbon dot fluorescence was tuned using an electron acceptor molecule. Our observation can make the carbon dots as an excellent candidate for the super-resolution imaging of nanoscale biomolecules within the cell.

Morphological effect of gold nanoparticles on the adsorption of bovine serum albumin.

Various properties of gold nanoparticles (GNPs) are found to play crucial roles in their biological activity. Among them, the morphology and surface chemistry are extremely important. This is because of differences in surface energies of various crystal facets arising from a large fraction of edges, corners and vertices. In the present work, we provide a comparative study on the adsorption and binding affinities of bovine serum albumin (BSA) onto triangular gold nanoplates (TGNP) and gold nanorods (GNR). The results were compared with similar size of both CTAB and citrate stabilized spherical GNPs. Our data suggested stronger binding of BSA on citrate stabilized spherical GNPs whereas TGNP shows the weakest binding among all the GNPs. A blue shift of approximately 20 nm in tryptophan fluorescence was observed for all CTAB stabilized GNPs, indicating the local dielectric changes surrounding the tryptophan residue. Loss of the secondary structure was also observed for all CTAB stabilized GNPs. No spectral shift was observed for citrate stabilized spherical GNPs though maximum quenching of fluorescence and minimum structural loss was observed. With the help of molecular simulation recently developed by our group, a binding model is proposed to explain all the above experimental results.

Fluorescence correlation spectroscopy at single molecule level on the Tat-TAR complex and its inhibitors.

The TAR element of HIV and the viral protein Tat form a molecular switch regulating transcriptional efficiency in HIV. We show that fluorescence correlation spectroscopy at the single molecule level is a powerful method to study the association between a Tat-derived peptide and TAR fragments. We also investigated the inhibition of the peptide-RNA complex by different ligands. Utilizing cross correlation measurements, the dissociation constants (K(D)) were determined. To demonstrate the important role of the bulge for the binding of Tat, we compared wt-TAR with three RNA mutants, mainly differing in the bulge region. For the TAR mutants studied at equimolar concentration of RNA and peptide (25 nM), the K(D) values are 15-35 times larger than that of wt-TAR. This gives evidence that the bulge region is the most crucial part of the TAR RNA for specific Tat binding. The IC(50) values for different inhibitors of the Tat/TAR complex both with wt-TAR and mutants have been determined. Neamine conjugate proved to be the best inhibitor of the complex formation. Our results are in agreement with earlier published data on this system using alternative biophysical and biochemical methods, respectively.