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Abstract: Polyhydroxy compounds are secondary metabolites that are ubiquitous in plants of high-er genera. They possess therapeutic properties against a wide spectrum of diseases, including can-cers, neurodegenerative disorders, atherosclerosis, as well as cardiovascular disease. The phyto-chemical flavonol (a type of flavonoid) kaempferol (KMP) (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4Hchromen-4-one) is abundant in cruciferous vegetables, including broccoli, kale, spinach, and wa-tercress, as well as in herbs like dill, chives, and tarragon. KMP is predominantly hydrophobic in nature due to its diphenylpropane structure (a characteristic feature of flavonoids). Recent findings have indicated the promise of applying KMP in disease prevention due to its potential antioxidant, antimutagenic, antifungal, and antiviral activities. In the literature, there is evidence that KMP ex-erts its anticancer effects by modulating critical elements in cellular signal transduction pathways linked to apoptosis, inflammation, angiogenesis, and metastasis in cancer cells without affecting the viability of normal cells. It has been shown that KMP triggers cancer cell death by several mecha-nisms, including cell cycle arrest, caspase activation, metabolic alteration, and impacting human te-lomerase reverse-transcriptase gene expression. This review is aimed at providing critical insights into the influence of KMP on the intracellular cascades that regulate metabolism and signaling in breast, ovarian, and cervical cancer cells.

Ovarian cancer is the deadliest gynecological cancer which rarely causes symptoms, and goes undetected until reaching the advanced stage of drug-resistant metastases. The cationic porphyrin meso-tetra(4-N-methylpyridyl)porphine (TMPyP) is a well-known photosensitizer (PS) used in photodyamic therapy (PDT) for curing cancer due to its strong affinity for DNA and high yield of reactive oxygen species (ROS) upon light activation. The practicality to irradiate tumor cells alone in the physiological system being slim (due to the close proximity of healthy cells and tumors), we looked for a variation in the PDT using a mixture of TMPyP with 1,5-dihydroxynapthalene (DHN) and Fe(III) ions at a mole ratio of 1:20:17 (drug combo) respectively in aqueous solution. The drug combo needs no photoactivation in H(2)O(2) rich environment (mimicking the microenvironment of cancer/tumor), where it generates ȮH and juglone, the latter being a known potent anticancer agent. In vitro studies of the drug combo in drug resistant and sensitive ovarian cancer cell lines showed drastic growth inhibition and cell death compared to normal epithelial cells. The drug combo provides an effective and non-invasive alternative to conventional PDT, exploiting the cytosolic carcinogenic H(2)O(2) to produce an efficient anticancer treatment. The unique action of cancer-specific cytotoxicity arises from the redox chemistry involving activation of Fe(III) as the oxidizing agent to generate juglone, which utilizes the cytosolic ROS in cancer cells against itself.

Background and Aims According to the World Health Organization (WHO), more than 75.7 million confirmed cases of coronavirus disease 2019 (COVID‐19), a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), have been reported so far. Researchers are working relentlessly to find effective solutions to this catastrophe, using genomic sequence‐based investigation, immunological analysis, and more. The role of health disparity has also emerged as an intriguing factor that made a huge impact on the lives of people. Methods We analyzed various factors that triggered the health disparity in the United States of America along with the rate of COVID‐19 morbidity and mortality. Furthermore, we have also focused on the State of Mississippi, which is suffering from an extreme health disparity. Data have been obtained from publicly available data sources including, Center for Disease Control and Prevention and Mississippi State Department of Health. Correlation analysis of the dataset has been performed using R software. Results Our analysis suggested that the COVID‐19 infection rate per 100 000 people is directly correlated with the increasing number of the African American population in the United States. We have found a strong correlation between the obesity and the COVID‐19 cases as well. All the counties in Mississippi demonstrate a strong correlation between a higher number of African American population to COVID‐19 cases and obesity. Our data also indicate that a higher number of African American populations are facing socioeconomic disadvantages, which enhance their chances of becoming vulnerable to pre‐existing ailments such as obesity, type‐2 diabetes, and cardiovascular diseases. Conclusion We proposed a possible explanation of increased COVID‐19 infectivity in the African American population in the United States. This work has highlighted the intriguing factors that increased the health disparity at the time of the COVID‐19 pandemic.

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.

Juicing vegetables is thought to be an anticancer treatment. Support exists for a rank order of anticancer greens (kale &gt, dandelion &gt, lettuce &gt, spinach) based on degrees of bioavailability of different phytochemicals, also offset by some noxious molecules (i.e., calcium-oxalate). We developed a new in vitro transepithelial anti-neuroblastoma model system. The juices were diluted as predicted once in the small intestine. They were applied to apical Caco-2Bbe1 cells atop dividing SH-SY5Y neuroblastoma cells, and changes in transepithelial electrical resistance (TEER) and cell growth were considered with juice spectroscopies. Studied first in monoculture, kale and dandelion were the most cytostatic juices on SH-SY5Ys, lettuce showed no effect, and high (4.2%) spinach was cytotoxic. In co-culture, high (4.2%) kale was quickest (three days) to inhibit neuroblastoma growth. By five days, dandelion and kale were equally robust. Lettuce showed small anti-proliferative effects at five days and spinach remained cytotoxic. Spinach’s cytotoxicity corresponded with major infrared bands indicative of oxalate. Kale juice uniquely induced reactive oxygen species and S-phase cell cycle arrest in SH-SY5Y. The superiority of kale and dandelion was also apparent on the epithelium, because raising TEER levels is considered healthy. Kale’s unique features corresponded with a major fluorescent peak that co-eluted with kaempferol during high performance liquid chromatography. Because the anticancer rank order was upheld, the model appears validated for screening anticancer juices.

We report the effectiveness of silver nanocluster (Ag-NC) against the biofilm of Pseudomonas aeruginosa (PA). Two DNA aptamers specific for PA and part of their sequences were chosen as templates for growing the Ag-NC. While circular dichroism (CD) studies determined the presence of secondary structures, UV/Vis absorption, and fluorescence spectroscopic studies confirmed the formation of the fluorescent Ag-NC on the DNA templates. Furthermore, mesoscopic physics-based partial wave spectroscopy (PWS) was used to analyze the backscattered light signal that can detect the degree of nanoscale mass density/refractive index fluctuations to identify the biofilm formation, comparatively among the different aptamers with respect to the control sample. The importance of the secondary structure of the aptamer DNA in targeting, successfully binding with the cells and delivering the Ag-NC, is evidenced by the decrease in disorder strength (Ld) of the Ag-NC treated samples compared to the untreated PA cells, which showed the abundance of higher Ld in the PWS studies. The higher Ld value attributed to the higher mass density fluctuations and the formation of biofilm. We envision this study to open a new avenue in using a powerful optical microscopic technique like PWS in detection, and DNA aptamer enclosed silver nanoclusters to prevent biofilms for opportunist pathogens like Pseudomonas aeruginosa.

Aniline, heterocyclic aromatic amines, and arylamines are known carcinogens. Recently aniline mustard has come into prominence as a novel anticancer agent. In this project, microwave irradiation has been used to synthesize an optically active alkylated aniline namely 2,6-dimethyl-4-(1-(p-tolyl)ethyl)aniline (abbreviated DMPA). The presence of quartet and doublet peaks in NMR and a single chromatogram in HPLC verified that the final product DMPA, prepared from the synthesis reactions, had no major impurities. By using a Lux chiral column in HPLC, two peaks have been detected in the chromatogram, which correspond to two enantiomers of the chiral aniline derivative. Fluorescence spectroscopic measurements on DMPA indicated conspicuous dependence of its emission behavior on the polarity (in terms of the empirical polarity parameter ET(30)) of the homogeneous solvents used, a property important for an optical sensor. The nature of the emission profiles, along with the relevant parameter namely wavelength at emission maximum (λemmax) is used to infer the distribution, binding and microenvironment of the DMPA molecules in human serum albumin protein (HSA). DMPA is weakly fluorescent in aqueous buffer medium, with a dramatic enhancement in the fluorescence emission in the presence of HSA. Molecular modeling studies have been carried out on the two enantiomers (R and S) of DMPA with HSA. The implications of these findings are examined in relation to the potentialities of DMPA as a novel fluorescence sensor for biological systems.

Small silver clusters that form with short oligonucleotides are distinguished by their strong fluorescence. Previous work showed that red and blue/green emitting species form with the cytosine oligonucleotide dC12. To understand how the bases and base sequence influence cluster formation, the blue/green emitting clusters that form with the thymine-containing oligonucleotides dT12, dT4C4T4, and dC4T4C4 are discussed. With dT12 and dT4C4T4, variations in the solution pH establish that the clusters associate with the N3 of thymine. The small clusters are bound to the larger DNA template, as demonstrated by fluorescence anisotropy, circular dichroism, and fluorescence correlation spectroscopy (FCS) studies. For dT4C4T4, FCS studies showed that approximately 50% of the strands are labeled with the fluorescent clusters. Absorption spectra and the gas dependence of the fluorescence show that nonfluorescent clusters also form following the reduction of the silver cation – oligonucleotide conjugates. Fluorescent cluster formation is favored by oxygen, thus indicating that the DNA-bound clusters are partially oxidized. To elaborate the sequence dependence of cluster formation, dC4T4C4 was studied. Cluster formation depends on the oligonucleotide concentration, and higher concentrations favor a red emitting species. A blue/green emissive species dominates at lower concentrations of dC4T4C4, and it has spectroscopic, physical, and chemical properties that are similar to those of the clusters that form with dT12 and dT4C4T4. These results suggest that cytosine- and thymine-containing oligonucleotides stabilize a preferred emissive silver cluster.

Fisetin (3,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) are the bioactive plant flavonoids that are potentially useful therapeutic drugs for the treatment of a broad spectrum of diseases, including atherosclerosis, cardiovascular disease, obesity, hypertension, and cancer. 3-Hydroxyflavone (3HF) and 7-hydroxyflavone (7HF) are the synthetic chromophores of fisetin and quercetin. We have exploited dual luminescence properties of fisetin and quercetin along with 3-HF and 7HF to examine their efficacy of binding and compare their interactions with DNA, which is one of the macromolecular targets of flavonoids in physiological systems. Following the sequence of the human telomeric DNA 5'-d (CCCTAA-)n/(-TTAGGG)n-5', two single-stranded DNA oligonucleotides, 5'-d(C3TA2)3C3-3' and 5'-d(T2AG3)4-3', and their duplex were used as receptors to study binding by the ligands quercetin, fisetin, and their chromophores. Circular dichroism, differential absorption, UV thermal melting, and size exclusion chromatographic studies indicated the formation of unusual DNA structures (such as C4 and G4 tetraplexes) for both the C- and G-rich single-stranded DNAs. Upon binding to DNA, dramatic changes were observed in the intrinsic fluorescence behavior of the flavonoids. Molecular docking studies were performed to describe the likely binding sites for the ligands. The spectroscopic studies on flavonoid-DNA interactions described herein demonstrate a powerful approach for examining their DNA binding through exploiting the highly sensitive intrinsic fluorescence properties of the flavonoids as their own "reporter" for their interactions with macromolecular targets.

In 1936, Rusznyak and Szent-Györgyi first drew attention to the therapeutically beneficial role of dietary flavonoids, which are the most common group of polyphenols ubiquitously present in plant based food and beverages. Recent years have witnessed a renascence of interest on these nutraceuticals, which, because of their high potency and low systemic toxicity, are gradually emerging as promising alternatives to conventional therapeutic drugs. There is a mounting evidence that various proteins frequently serve as targets for therapeutically important flavonoids. In this article we present perspectives exemplifying the growing potential of fluorescence spectroscopy as an exquisitely sensitive tool for noninvasive sensing of protein-flavonoid interactions at physiologically relevant concentrations, via measurements of steady state emission parameters as well as decay kinetics studies of the intrinsic fluorescence of the target (protein) and/or ligand (flavonoid). Especially, we highlight novel applications of the remarkably environment sensitive 'two color' fluorescence exhibited by many important flavonoids, which permits multiparametric and ratiometric measurements. To consolidate findings obtained via fluorescence spectroscopy, use of other relevant experimental biophysical techniques and molecular modeling have proved to be valuable and are also discussed here. Such complementary studies provide additional insights regarding the thermodynamics and conformational aspects of the protein-flavonoid interactions, together with details, at atomistic level, of the dominant noncovalent interactions involved in the docking of different flavonoids to their target proteins.

Steady state and time resolved fluorescence spectroscopy, combined with molecular dynamics simulation, have been used to explore the interactions of a therapeutically important bioflavonoid, genistein, with normal human hemoglobin (HbA). Binding constants estimated from the fluorescence studies were K = (3.5  0.32)  10 4 M −1 for genistein. Specific interactions with HbA were confirmed from flavonoid-induced fluorescence quenching of the tryptophan in the protein HbA. The mechanism of this quenching involves both static and dynamic components as indicated by: (a) increase in the values of Stern-Volmer quenching constants with temperatures, (b) 0  / is slightly > 1 (where 0  and  are the unquenched and quenched tryptophan fluorescence lifetimes (averaged) respectively). Molecular docking and dynamic simulations reveal that genistein binds between the subunits of HbA, ~18 A away from the closest heme group of chain α1, emphasizing the fact that the drug does not interfere with oxygen binding site of HbA.

Photostability, inherent fluorescence brightness, and optical modulation of fluorescence are key attributes distinguishing silver nanoclusters as fluorophores. DNA plays a central role both by protecting the clusters in aqueous environments and by directing their formation. Herein, we characterize a new near-infrared-emitting cluster with excitation and emission maxima at 750 and 810 nm, respectively, that is stabilized within C3AC3AC3TC3A. Following chromatographic resolution of the near-infrared species, a stoichiometry of 10 Ag/oligonucleotide was determined. Combined with excellent photostability, the cluster’s 30% fluorescence quantum yield and 180 000 M−1 cm−1 extinction coefficient give it a fluorescence brightness that significantly improves on that of the organic dye Cy7. Fluorescence correlation analysis shows an optically accessible dark state that can be directly depopulated with longer wavelength coillumination. The coupled increase in total fluorescence demonstrates that enhanced sensitivity...

When compared with silver nanoparticles, silver clusters comprised of ∼101 atoms are distinguished by their strong fluorescence, and DNA directs and stabilizes particular types of clusters via base-specific interactions. Two main observations considered in this paper are the pH dependence of the fluorescence and the folded conformation of the oligonucleotide−cluster conjugates. Two i-motif forming oligonucleotides (dTA2C4)4 and (dC4A2)3C4 coordinate red and green emissive species, and these fluorescent species are favored in slightly acidic and basic solutions, respectively. The red emission is highest at pH 6, at which the i-motif forms of the oligonucleotides are also stable. When assessed by size exclusion chromatography, the oligonucleotide and cluster conjugate have similar global structures, which indicate that the DNA strands are similarly organized at this pH. The green emission is highest at pH 8−9. In these basic solutions, the oligonucleotide alone is unfolded, yet the green and red cluster−oli...

Some years ago the non-natural amino acid 5-hydroxytryptophan (5HT) was proposed as a novel intrinsic fluorescence probe for protein structure, function and dynamics. In this connection, 5-hydroxyindole (5HI) merits particular attention, since it is the chromophoric moiety of 5HT. In our previous reports it was shown that unlike other well-known fluorescence probes, the fluorescence emission maximum ( λ em max ) of the 5HI chromophore is insensitive to environment. Here we demonstrate the potential utility of 5HI as an extrinsic optical probe for biological systems (such as membranes and proteins), exploiting its fluorescence anisotropy ( r ) as a useful parameter. Small, unilamellar liposomal membranes of synthetic phosphatidylcholine, (DPPC) and the protein bovine serum albumin (BSA) were chosen as testing grounds. Our studies reveal that the anisotropy ( r ) of 5HI serves as an excellent monitor for estimating the gel to liquid crystalline phase transition temperature ( T m ) of the phospholipid, as well as protein binding characteristics (binding constant and Gibbs free energy). Implications of these results are discussed.

5-Hydroxytryptophan (5HT) has been the focus of much recent attention as a novel intrinsic fluorescence probe for proteins. Solvent-dependence studies reported here show that, compared to other fluorophores of related interest like tryptophan (Trp) and 7-azatryptophan (7AT), the fluorescence emission maximum (λemmax) of 5HT is relatively insensitive to solvent polarity. Thus, upon change of solvent from acetonitrile to water, the λemmax of 5HT fluorescence is red shifted by only 3 nm, in contrast to corresponding red shifts of much larger magnitudes observed for Trp and 7AT (14 and 23 nm, respectively). This behaviour suggests the lack of significant solvent dipolar relaxation effects in 5HT, which is corroborated by the low temperature emission measurements at 77°K. This aspect is also revealed in the fluorescence characteristics of 5HT in a membrane-mimetic model system, namely in AOT reverse-micellar assemblies containing different amounts of water. With increase in water content, the λemmax of 5HT remains almost unaffected, although fluorescence anisotropy and quenching data clearly indicate significant changes in the microenvironments of 5HT molecules.

Quadruplex (G4) forming sequences in telomeric DNA and c-myc promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G4 can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) in a G4 DNA matrix. The excited state intra-molecular proton transfer (ESPT) of fisetin plays an important role in observing and understanding the binding of fisetin with the G4 DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G4 DNA and size exclusion chromatography (SEC) proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G4 ligand.

Fluorescent silver nanoclusters (Ag-NCs) are in prominence as novel sensing materials due to their biocompatibility, photostability, and molecule-like optical properties. The present work is carried out on an array (17 sequences) of 16 bases long cytosine rich, single stranded DNA templates 5′-C3XiC3XiiC3XiiiC3Xiv-3′ where i, ii, iii, iv correspond to T/G/C deoxynucleobases (with default base A). Among all the oligonucleotides, a sequence C3AC3AC3TC3G (3T4G) has been identified, which grows three different near-infrared-emitting NC species with absorption/emission maxima at ~620/700 (species I), 730/800 (species II), and 830 (Species III) nm, respectively. The nature of the spectral profiles, along with relevant parameters namely absorption maximum (\(\lambda_{abs}^{max}\)), emission maximum (\(\lambda_{em}^{max}\)), anisotropy (r), lifetime (\(\tau\)), circular dichroism spectral data are used to understand the microenvironments of the fluorescent NC species I, II, and III. DNA:Ag stiochiometric, pH and solvent dependent studies proved that i-motif scaffolds with different folding topologies are associated with the growth of these three species and a certain concentration of silver and H+ favor the growth of species III. Size exclusion chromatographic measurements provided similar indications that a folded, more compact, classic i-motif template is associated with the formation of the longer NIR (~830 nm) absorbing species. This study provides a more definitive approach to design and obtain a targeted DNA templated Ag-NC with required emission properties for biophysical and cellular applications.

Steady state and time resolved fluorescence along with anisotropy and induced circular dichroism (ICD) spectroscopy provide useful tools to observe and understand the behavior of the therapeutically important plant flavonoids fisetin and daidzein in γ-cyclodextrin (γ-CDx) nanocavity. Benesi–Hildebrand plots indicated 1:1 stoichiometry for both the supramolecular complexes. However, the mode of the binding of fisetin significantly differs from daidzein in γ-CDx, as is observed from ICD spectra which is further confirmed by docking studies. The interaction with γ-CDx proceeds mainly by the phenyl ring and partly by the chromone ring of fisetin whereas only the phenyl ring takes part for daidzein. A linear increase in the aqueous solubility of the flavonoids is assessed from the increase in the binding of the flavonoids with the γ-CDx cavity, which are determined by the gradual increase in the ICD signal, fluorescence emission as well as increase in fluorescence anisotropy with increasing (γ-CDx). This confirms γ-CDx as a nanovehicle for the flavonoids fisetin and daidzein in improving their bioavailability.

Hesperitin, a ubiquitous bioactive flavonoid abundant in citrus fruits is known to possess antioxidant, anti-carcinogenic, hypolipidemic, vasoprotective and other important therapeutic properties. Here we have explored the interactions of hesperitin with normal human hemoglobin (HbA), using steady state and time resolved fluorescence spectroscopy, far UV circular dicroism (CD) spectroscopy, combined with molecular modeling computations. Specific interaction of the flavonoid with HbA is confirmed from flavonoid-induced static quenching which is evident from steady state fluorescence as well as lifetime data. Both temperature dependent fluorescence measurements and molecular docking studies reveal that apart from hydrogen bonding and van der Waals interactions, electrostatic interactions also play crucial role in hesperitin-HbA interactions. Furthermore, electrostatic surface potential calculations indicate that the hesperitin binding site in HbA is intensely positive due to the presence of several lysine and histidine residues.

The present study establishes the effectiveness of natural drug delivery mechanisms and investigates the interactions between drug and its natural carrier. The binding between the isoflavone diadzein (DZN) and the natural carrier hemoglobin (HbA) was studied using optical spectroscopy and molecular dynamics simulations. The inherent fluorescence emission characteristics of DZN along with that of tryptophan (Trp) residues of the protein HbA were exploited to elucidate the binding location and other relevant parameters of the drug inside its delivery vehicle HbA. Stern-Volmer studies at different temperatures indicate that static along with collisional quenching mechanisms are responsible for the quenching of protein fluorescence by the drug. Molecular dynamics and docking studies supported the hydrophobic interactions between ligand and protein, as was observed from spectroscopy. DZN binds between the subunits of HbA, ∼15 A away from the closest heme group of chain α1, emphasizing the fact that the drug does not interfere with oxygen binding site of HbA.

Intrinsic dynamics of DNA plays a crucial role in DNA protein interactions and has been emphasized as a possible key component for in vivo chromatin organization. We have prepared an entangled DNA microtube above the overlap concentration by exploiting the complementary cohesive ends of lambda-phage DNA, which is confirmed by atomic force microscopy and agarose gel electrophoresis. Photon correlation spectroscopy further confirmed that the entangled solutions are found to exhibit the classical hydrodynamics of a single chain segment on length scales smaller than the hydrodynamic length scale of single lambda-phage DNA molecule. We also observed that in 41.6% (gm water/gm DNA) hydrated state, lambda-phage DNA exhibits a dynamic transition temperature (T-dt) at 187 K and a crossover temperature (T-c) at 246 K. Computational insight reveals that the observed structure and dynamics of entangled lambda-phage DNA are distinctively different from the behavior of the corresponding unentangled DNA with open cohesive ends, which is reminiscent with our experimental observation.

Bifunctional DNA oligonucleotides serve as templates for chromophoric silver clusters and as recognition sites for target DNA strands, and communication between these two components is the basis of an oligonucleotide sensor. Few-atom silver clusters exhibit distinct electronic spectra spanning the visible and near-infrared region, and they are selectively synthesized by varying the base sequence of the DNA template. In these studies, a 16-base cluster template is adjoined with a 12-base sequence complementary to the target analyte, and hybridization induces structural changes in the composite sensor that direct the conversion between two spectrally and stoichiometrically distinct clusters. Without its complement, the sensor strand selectively harbors ~7 silver atoms that absorb at 400 nm and that fold the DNA host. Upon association of the target with its recognition site, the sensor strand opens to expose the cluster template that has the binding site for ~11 silver atoms, and absorption at 720 nm with relatively strong emission develops in lieu of the violet absorption. Variations in the length and composition of the recognition site and the cluster template indicate that these types of dual component sensors provide a general platform for near infrared-based detection of oligonucleotides in challenging biological environments.

Plant flavonoids are emerging as novel therapeutic drugs for free radical mediated diseases, for which cell membranes mainly serve as targets for lipid peroxidation and related deleterious effects. Screening and characterization of these ubiquitous, therapeutically potent polyphenolic compounds require a clear understanding regarding their binding and possible locations in membranes, as well as quantitative estimates of relevant parameters such as partition coefficients, antioxidant and radical scavenging capacities. In this article we present perspectives emphasizing novel uses of the exquisitely sensitive ‘two color’ intrinsic fluorescence of plant flavonoids (which arise due to highly efficient photoinduced excited state intramolecular proton transfer (ESIPT) reactions) to explore their binding to model biomembranes consisting of phosphatidylcholine liposomes. Extension of such studies to natural biomembranes of relevant interest is also exemplified. Spectrophotometric assays reveal that typical mono- as well as poly-hydroxy substituted flavonoids have remarkable inhibitory actions on lipid peroxidation, and are significantly more potent antioxidants (2.5–4 times higher) compared to the reference compound Trolox (an water soluble derivative of vitamin E). The structure–activity relationships emerging from such studies are consistent with theoretical predictions based on quantum chemical computations.

A bifunctional oligonucleotide integrates in situ synthesis of a fluorogenic silver cluster with recognition of a target DNA sequence. With the template C(3)AC(3)AC(3)GC(3)A, a complex forms with 10 silver atoms that possesses electronic transitions in the near-infrared and that is detected at nanomolar concentrations using diode laser excitation. Pendant to this cluster encoding region, the recognition component binds a target DNA strand through hybridization, and decoupling of these two regions of the composite sensor renders a modular sensor for specific oligonucleotides. A target is detected using a quencher strand that bridges the cluster template and recognition components and disturbs cluster binding, as indicated by static quenching. Competitive displacement of the quencher by the target strand restores the favored cluster environment, and our key finding is that this exchange enhances emission through a proportional increase in the number of emissive clusters. DNA detection is also accomplished in serum-containing buffers by taking advantage of the high brightness of this fluorophore and the inherently low endogenous background in the near-infrared spectral region. Cluster stability in this biological environment is enhanced by supplementing the solutions with Ag(+).

Few-atom silver clusters harbored by DNA are promising fluorophores due to their high molecular brightness along with their long- and short-term photostability. Furthermore, their emission rate can be enhanced when co-illuminated with low-energy light that optically depopulates the fluorescence-limiting dark state. The photophysical basis for this effect is evaluated for two near-infrared-emitting clusters. Clusters emitting at ∼800 nm form with C(3)AC(3)AC(3)TC(3)A and C(3)AC(3)AC(3)GC(3)A, and both exhibit a trap state with λ(max) ∼ 840 nm and an absorption cross section of (5-6) × 10(-16) cm(2)/molecule that can be optically depopulated. Transient absorption spectra, complemented by fluorescence correlation spectroscopy studies, show that the dark state has an inherent lifetime of 3-4 μs and that absorption from this state is accompanied by photoinduced crossover back to the emissive manifold of states with an action cross section of ∼2 × 10(-18) cm(2)/molecule. Relative to C(3)AC(3)AC(3)TC(3)A, C(3)AC(3)AC(3)GC(3)A produces a longer-lived trap state and permits more facile passage back to the emissive manifold. With the C(3)AC(3)AC(3)AC(3)G template, a spectrally distinct cluster forms having emission at ∼900 nm, and its trap state has a ∼4-fold shorter lifetime. These studies of optically gated fluorescence bolster the critical role of the nucleobases in both the formation and excited state dynamics of these highly emissive metallic clusters.

Photostability, inherent fluorescence brightness, and optical modulation of fluorescence are key attributes distinguishing silver nanoclusters as fluorophores. DNA plays a central role both by protecting the clusters in aqueous environments and by directing their formation. Herein, we characterize a new near infrared-emitting cluster with excitation and emission maxima at 750 and 810 nm, respectively that is stabilized within C(3)AC(3)AC(3)TC(3)A. Following chromatographic resolution of the near infrared species, a stoichiometry of 10 Ag/oligonucleotide was determined. Combined with excellent photostability, the cluster's 30% fluorescence quantum yield and 180,000 M(-1)cm(-1) extinction coefficient give it a fluorescence brightness that significantly improves on that of the organic dye Cy7. Fluorescence correlation analysis shows an optically accessible dark state that can be directly depopulated with longer wavelength co-illumination. The coupled increase in total fluorescence demonstrates that enhanced sensitivity can be realized through Synchronously Amplified Fluorescence Image Recovery (SAFIRe), which further differentiates this new fluorophore.

Long repeated sequences of DNA and their associated secondary structure govern the development and severity of a significant class of neurological diseases. Utilizing the effect of base stacking on fluorescence quantum yield, 2-aminopurine substitutions for adenine previously demonstrated sequestered bases in the stem and exposed bases in the loop for an isolated (CAG) 8 sequence. This study evaluates (CAG) 8 that is incorporated into a duplex, as this three-way junction is a relevant model for intermediates that lead to repeat expansion during DNA replication and repair. From an energetic perspective, thermally induced denaturation indicates that the duplex arms dictate stability and that the secondary structure of the repeated sequence is disrupted. Substitutions with 2-aminopurine probe base exposure throughout this structure, and two conclusions about secondary structure are derived. First, the central region of (CAG) 8 is more solvent-exposed than single-stranded DNA, which suggests that hairpin formation in the repeated sequence is disrupted. Second, base stacking becomes compromised in the transition from the duplex to (CAG) 8 , resulting in bases that are most similar to single-stranded DNA at the junction. Thus, an open (CAG) 8 loop and exposed bases in the arms indicate that the strand junction profoundly influences repeated sequences within three-way junctions.

Polymorphic forms of nucleic acids provide platforms for new nanomaterials, and transition metal cations give access to alternative arrangements of nucleobases by coordinating with electron-rich functional groups. Interaction of Ag(+) with 5'-guanosine monophosphate (5'-GMP) is considered in this work. Ag(+) promotes nucleotide stacking and aggregation, as indicated by the increased viscosity of 5'-GMP solutions with Ag(+), magnification of the circular dichroism response of guanine by Ag(+), and exothermic reactions between Ag(+) and guanine derivatives. Isothermal titration calorimetry studies show that the reaction is favored starting at 10 microM 5'-GMP. Utilizing the exothermic heat change associated with reaction of Ag(+) with 5'-GMP, local structure within the aggregate was assessed. On the basis of the salt dependence of the reaction and comparison with the corresponding nucleoside, the dianionic phosphate of 5'-GMP is one binding site for Ag(+), although this electrostatic interaction is not a dominant contribution to the overall heat change. Another binding site is the N7 on the nucleobase, as determined via studies with 7-deazaguanosine. Besides this binding site, Ag(+) also associates with the O6, as earlier studies deduced from the shift in the carbonyl stretching frequency associated with adduct formation. With these two binding sites on the nucleobase, the empirical stoichiometry of approximately 1 Ag(+):nucleobase derived from the calorimetry studies indicates that Ag(+) coordinates two nucleobases. The proposed structural model is a Ag(+)-mediated guanine dimer within a base stacked aggregate.

Recent years have witnessed burgeoning interest in plant flavonoids as novel therapeutic drugs targeting cellular membranes and proteins. Motivated by this scenario, we explored the binding of robinetin (3,7,3′,4′,5′-pentahydroxyflavone, a bioflavonoid with remarkable ‘two color’ intrinsic fluorescence properties), with egg yolk phosphatidylcholine (EYPC) liposomes and normal human hemoglobin (HbA), using steady state and time resolved fluorescence spectroscopy. Distinctive fluorescence signatures obtained for robinetin indicate its partitioning ( K p = 8.65 × 10 4 ) into the hydrophobic core of the membrane lipid bilayer. HbA–robinetin interaction was examined using both robinetin fluorescence and flavonoid-induced quenching of the protein tryptophan fluorescence. Specific interaction with HbA was confirmed from three lines of evidence: (a) bimolecular quenching constant K q ≫ diffusion controlled limit; (b) closely matched values of Stern–Volmer quenching constant and binding constant; (c) τ 0 / τ = 1 (where τ 0 and τ are the unquenched and quenched tryptophan fluorescence lifetimes, respectively). Absorption spectrophotometric assays reveal that robinetin inhibits EYPC membrane lipid peroxidation and HbA glycosylation with high efficiency.

The secondary structure of repeated trinucleotide sequences results in the development of several neurodegenerative diseases, and these studies consider the (CAG)(8) sequence that forms a stem-loop hairpin. The structural and thermodynamic properties of this hairpin are assessed using 2-aminopurine substitutions for adenine at six positions in this repeated sequence. Circular dichroism spectra and thermal denaturation experiments show that the secondary structure is not disturbed by the modifications. The local structure of the hairpin was monitored using the fluorescence intensities of 2-aminopurines, the changes in the intensity relative to the denatured state, and the sensitivity of the fluorescence to quenching by acrylamide. To establish the stem and loop characteristics in (CAG)(8), known reference points for stem, loop, and exposed base motifs were used. In the vicinity of the loop, the bases become more solvent exposed, which suggests that the instability associated with this repeated hairpin influences the global secondary structure. These results provide the basis to interpret the structures adopted by other repeated (CAG) structures.

Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3-hydroxyflavone (3HF), using the excited-state proton-transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from lambda(max)(em) = 513 nm to lambda(max)(em) = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion-restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA-3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 - 1.3 x 10(5) M(-1) for both these sites. Proteins 2001;43:75-81.