Fluorescence-quenching Assay Research Articles

Sinigrin reduces the virulence of Staphylococcus aureus by targeting coagulase

Multi-resistant Staphylococcus aureus (S. aureus) infection is a significant global health concern owing to its high mortality and morbidity rates. Coagulase (Coa), a key enzyme that activates prothrombin to initiate host coagulation, has emerged as a promising target for anti-infective therapeutic approaches. This study identified sinigrin as a potent Coa inhibitor that significantly inhibited S. aureus-induced coagulation at concentration as low as 32 mg/L. Additionally, at a higher concentration of 128 mg/L, sinigrin disrupted the self-protection mechanism of S. aureus. Thermal shift and fluorescence-quenching assays confirmed the direct binding of sinigrin to the Coa protein. Molecular docking analysis predicted specific binding sites for sinigrin in the Coa molecule, and point mutation experiments highlighted the importance of Arg-187 and Asp-222 as critical binding sites for both Coa and sinigrin. In vivo studies demonstrated that the combination of sinigrin with oxacillin exhibited greater antibacterial efficacy than oxacillin alone in the treatment of S. aureus-induced pneumonia in mice. Furthermore, sinigrin was shown to reduce bacterial counts and inflammatory cytokine levels in the lung tissues of S. aureus-infected mice. In summary, sinigrin was shown to directly target Coa, resulting in the attenuation of S. aureus virulence, which suggests the potential of sinigrin as an adjuvant for future antimicrobial therapies.

The use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains.

The natural asymmetry of the lipid bilayer in biological membranes is, in part, a testament to the complexity of the structure and function of this barrier limiting and protecting cells (or organelles). These lipid bilayers consist of two lipid leaflets with different lipid compositions, resulting in unique interactions within each leaflet. These interactions, combined with interactions between the two leaflets, determine the overall behavior of the membrane. Model membranes provide the most suitable option for investigating the fundamental interactions of lipids. This report describes a comprehensive method to make asymmetric giant unilamellar vesicles (aGUVs) using the technique of hemifusion. In this method, calcium ions induce the hemifusion of giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB), both having different lipid compositions. During hemifusion, a stalk, or a more commonly seen hemifusion diaphragm, connects the outer leaflets of GUVs and the SLB. The lateral diffusion of lipids naturally promotes the lipid exchange between the connected outer leaflets. After calcium chelation to prevent further fusion, a mechanical shear detaches aGUVs from the SLB. A fluorescence quench assay is employed to test the extent of bilayer asymmetry. A fluorescence quenching assay tests bilayer asymmetry and verifies dye and lipid migration to a GUV's outer leaflet.

Lyso-DGTS Lipid Derivatives Enhance PON1 Activities and Prevent Oxidation of LDL: A Structure-Activity Relationship Study.

Paraoxonase 1 (PON1) plays a role in regulating reverse cholesterol transport and has antioxidative, anti-inflammatory, antiapoptotic, vasodilative, and antithrombotic activities. Scientists are currently focused on the modulation of PON1 expression using different pharmacological, nutritional, and lifestyle approaches. We previously isolated a novel active compound from Nannochloropsis microalgae—lyso-diacylglyceryltrimethylhomoserine (lyso-DGTS)—which increased PON1 activity, HDL-cholesterol efflux, and endothelial nitric oxide release. Here, to explore this important lipid moiety’s effect on PON1 activities, we examined the effect of synthesized lipid derivatives and endogenous analogs of lyso-DGTS on PON1 lactonase and arylesterase activities and LDL oxidation using structure–activity relationship (SAR) methods. Six lipids significantly elevated recombinant PON1 (rePON1) lactonase activity in a dose-dependent manner, and four lipids significantly increased rePON1 arylesterase activity. Using tryptophan fluorescence-quenching assay and a molecular docking method, lipid–PON1 interactions were characterized. An inverse correlation was obtained between the lactonase activity of PON1 and the docking energy of the lipid–PON1 complex. Furthermore, five of the lipids increased the LDL oxidation lag time and inhibited its propagation. Our findings suggest a beneficial effect of lyso-DGTS or lyso-DGTS derivatives through increased PON1 activity and prevention of LDL oxidation.

Delineating the activity of the potent nicotinic acetylcholine receptor agonists (+)-anatoxin-a and (-)-hosieine-A.

The affinity and thermodynamic parameters for the interactions of two naturally occurring neurotoxins, (+)-anatoxin-a and (-)-hosieine-A, with acetylcholine-binding protein were investigated using a fluorescence-quenching assay and isothermal titration calorimetry. The crystal structures of their complexes with acetylcholine-binding protein from Aplysia californica (AcAChBP) were determined and reveal details of molecular recognition in the orthosteric binding site. Comparisons treating AcAChBP as a surrogate for human α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the molecular features involved in ligand recognition and affinity for the protein targets are conserved. The ligands exploit interactions with similar residues as the archetypal nAChR agonist nicotine, but with greater affinity. (-)-Hosieine-A in particular has a high affinity for AcAChBP driven by a favorable entropic contribution to binding. The ligand affinities help to rationalize the potent biological activity of these alkaloids. The structural data, together with comparisons with related molecules, suggest that there may be opportunities to extend the hosieine-A scaffold to incorporate new interactions with the complementary side of the orthosteric binding site. Such a strategy may guide the design of new entities to target human α4β2 nAChR that may have therapeutic benefit.

Mammalian derived lipocalin and secretoglobin respiratory allergens strongly bind ligands with potentially immune modulating properties.

Allergens from furry animals frequently cause sensitization and respiratory allergic diseases. Most relevant mammalian respiratory allergens belong either to the protein family of lipocalins or secretoglobins. Their mechanism of sensitization remains largely unresolved. Mammalian lipocalin and secretoglobin allergens are associated with a function in chemical communication that involves abundant secretion into the environment, high stability and the ability to transport small volatile compounds. These properties are likely to contribute concomitantly to their allergenic potential. In this study, we aim to further elucidate the physiological function of lipocalin and secretoglobin allergens and link it to their sensitizing capacity, by analyzing their ligand-binding characteristics. We produced eight major mammalian respiratory allergens from four pet species in E.coli and compared their ligand-binding affinities to forty-nine ligands of different chemical classes by using a fluorescence-quenching assay. Furthermore, we solved the crystal-structure of the major guinea pig allergen Cav p 1, a typical lipocalin. Recombinant lipocalin and secretoglobin allergens are of high thermal stability with melting temperatures ranging from 65 to 90°C and strongly bind ligands with dissociation constants in the low micromolar range, particularly fatty acids, fatty alcohols and the terpene alcohol farnesol, that are associated with potential semiochemical and/or immune-modulating functions. Through the systematic screening of respiratory mammalian lipocalin and secretoglobin allergens with a large panel of potential ligands, we observed that total amino acid composition, as well as cavity shape and volume direct affinities to ligands of different chemical classes. Therefore, we were able to categorize lipocalin allergens over their ligand-binding profile into three sub-groups of a lipocalin clade that is associated with functions in chemical communication, thus strengthening the function of major mammalian respiratory allergens as semiochemical carriers. The promiscuous binding capability of hydrophobic ligands from environmental sources warrants further investigation regarding their impact on a molecule's allergenicity.

Nimodipine inhibits intestinal and aortic smooth muscle contraction by regulating Ca2+-activated Cl− channels

Nimodipine is a clinically used dihydropyridine L-type calcium channel antagonist that effectively inhibits transmembrane Ca2+ influx following the depolarization of smooth muscle cells, but the detailed effect on smooth muscle contraction is not fully understood. Ca2+-activated Cl− channels (CaCCs) in vascular smooth muscle cells (VSMCs) may regulate vascular contractility. We found that nimodipine can inhibit transmembrane protein 16A (TMEM16A) activity in a concentration-dependent manner by cell-based fluorescence-quenching assay and short-circuit current analysis, with an IC50 value of ~5 μM. Short-circuit current analysis also showed that nimodipine prevented Ca2+-activated Cl− current in both HT-29 cells and mouse colonic epithelia accompanied by significantly decreased cytoplasmic Ca2+ concentrations. In the absence of extracellular Ca2+, nimodipine still exhibited an inhibitory effect on TMEM16A/CaCCs. Additionally, the application of nimodipine to CFTR-expressing FRT cells and mouse colonic mucosa resulted in mild activation of CFTR-mediated Cl− currents. Nimodipine inhibited basolateral CCh-activated K+ channel activity with no effect on Na+/K+-ATPase activity. Evaluation of intestinal smooth muscle contraction showed that nimodipine inhibits intestinal smooth muscle contractility and frequency, with an activity pattern that was similar to that of non-specific inhibitors of CaCCs. In aortic smooth muscle, the expression of TMEM16A in thoracic aorta is higher than that in abdominal aorta, corresponding to stronger maximum contractility in thoracic aorta smooth muscle stimulated by phenylephrine (PE) and Eact. Nimodipine completely inhibited the contraction of aortic smooth muscle stimulated by Eact, and partially inhibited the contraction stimulated by PE. In summary, the results indicate that nimodipine effectively inhibits TMEM16A/CaCCs by reduction transmembrane Ca2+ influx and directly interacting with TMEM16A, explaining the mechanisms of nimodipine relaxation of intestinal and aortic smooth muscle contraction and providing new targets for pharmacological applications.

Multistep substrate binding and engagement by the AAA+ ClpXP protease

Escherichia coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.

Discovery of the Novel Inhibitor Against New Delhi Metallo-β-Lactamase Based on Virtual Screening and Molecular Modelling.

New Delhi metallo-β-lactamase (NDM-1), one of the metallo-β-lactamases (MBLs), leads to antibiotic resistance in clinical treatments due to the strong ability of hydrolysis to almost all kinds of β-lactam antibiotics. Therefore, there is the urgent need for the research and development of the novel drug-resistant inhibitors targeting NDM-1. In this study, ZINC05683641 was screened as potential NDM-1 inhibitor by virtual screening and the inhibitor mechanism of this compound was explored based on molecular dynamics simulation. The nitrocefin assay showed that the IC50 value of ZINC05683641 was 13.59 ± 0.52 μM, indicating that the hydrolytic activity of NDM-1 can be obviously suppressed by ZINC05683641. Further, the binding mode of ZINC05683641 with NDM-1 was obtained by molecular modeling, binding free energy calculation, mutagenesis assays and fluorescence-quenching assays. As results, ILE-35, MET-67, VAL-73, TRP-93, CYS-208, ASN-220 and HIS-250 played the key roles in the binding of NDM-1 with ZINC05683641. Interestingly, these key residues were exactly located in the catalytic activity region of NDM-1, implying that the inhibitor mechanism of ZINC05683641 against NDM-1 was the competitive inhibition. These findings will provide an available approach to research and develop new drug against NDM-1 and treatment for bacterial resistance.

A Structural Rationale for N-Methylbicuculline Acting as a Promiscuous Competitive Antagonist of Inhibitory Pentameric Ligand-Gated Ion Channels.

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABAA receptors and affects other pentameric ligand-gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence-quenching assay and isothermal titration calorimetry to record low-micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine-binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Å resolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABAA receptor-bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures, but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand-gated ion channels in the CNS.

Plumbagin Prevents Secretory Diarrhea by Inhibiting CaCC and CFTR Channel Activities.

Secretory diarrhea, which primarily originates through intestinal pathogens and viruses, is a health burden in many regions worldwide. Enterocyte Cl− channels, as the final step in enterotoxin-induced fluid secretion, constitute an attractive class of targets for diarrhea therapy. Chloride channel inhibitors have become a new class of candidates for antisecretion and anti-intestinal motility agents. In the present study, we identified plumbagin as a transmembrane protein 16A (TMEM16A) inhibitor in a cell-based fluorescence-quenching assay, and the IC50 value was ∼12.46 µM. Short-circuit current measurements showed that plumbagin reversibly inhibited the Eact-induced Cl− current on the apical side of TMEM16A-transfected Fischer rat thyroid (FRT) cells with no significant effect on cytoplasmic Ca2+ signaling. Notably, plumbagin also inhibited the activity of intestinal epithelial calcium-activated chloride channel (CaCC) and cystic fibrosis transmembrane conductance regulator (CFTR) in both HT-29 cells and mouse colons, but had no effects on the activity of the Na+-K+ ATPase or K+ channels. In in vivo experiments, the administration of plumbagin reduced both Escherichia coli heat-stable enterotoxin (STa)- and cholera toxin (CT)-induced intestinal fluid secretion. In neonatal mouse models of CT- and rotavirus infection-induced diarrhea, 0.4 µg plumbagin inhibited secretory diarrhea by >40% and 50%, respectively, without affecting intestinal epithelial integrity or the rotaviral infection. In addition, plumbagin exerted inhibitory effects on the vasoactive intestinal peptide (VIP)-, prostaglandin E2 (PGE2)-, and 5-hydroxytryptamine (5-HT)-stimulated Cl− currents. In the evaluations of intestinal motility, plumbagin significantly delayed intestinal motility and inhibited intestinal smooth muscle contractility without an evident impact on contractive frequency. Collectively, our results indicate that plumbagin inhibits both Ca2+- and cAMP-activated Cl− channels, accounting for the mechanisms of plumbagin inhibition of chloride secretion and intestinal motility. Thus, plumbagin can be a lead compound in the treatment of CT-induced, Traveler’s, and rotaviral diarrhea, as well as other types of secretory diarrhea that result from excessive intestinal fluid secretion and increased intestinal peristalsis.

Design and Synthesis of WM5 Analogues as HIV-1 TAR RNA Binders

Background: The 6-aminoquinolone WM5, previously identified by us, is among the most selective small molecules known as TAR RNA binders to show anti-HIV activity. Methods: Starting from WM5, a series of analogues modified at N-1, C-6 or C-7 position was prepared by inserting guanidine or amidine groups as well as other protonable moieties intended to electrostatically bind the phosphate backbone of TAR. All the compounds were tested for their ability to inhibit HIV-1 replication in MT-4 cells and in parallel for their cytotoxicity. The active compounds were also evaluated for their ability to interfere with the formation of the Tat-TAR complex using a Fluorescence Quenching Assay (FQA). Results: Some of the synthesized compounds showed an anti-HIV-1 activity in the sub-micromolar range with the naphthyridone derivatives being the most potent. Three of the synthesized derivatives were able to interact with the Tat-TAR complex formation presenting Ki values improved as compared to the values obtained with WM5. Conclusion: The addition of a pyridine-based protonable side chain at the N-1 position of the quinolone/naphthyridone core imparted to the compounds the ability to interfere with Tat-TAR complex formation and HIV-1 replication.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 μM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Investigation of the inhibition effect and mechanism of myricetin to Suilysin by molecular modeling

In the present study, the inhibitory effect and mechanism of myricetin, a natural flavonoid compound, in relation to Suilysin (SLY) were investigated through molecular dynamics simulations, mutational analysis and fluorescence-quenching assays. Myricetin is a potential inhibitor that does not exhibit antimicrobial activity but has been shown to inhibit SLY cytotoxicity. Molecular dynamics simulations and mutational analysis revealed that myricetin binds directly to SLY in the gap between domains 2 and 3, an important region for oligomerization and pore formation. The results of principal component analysis (PCA) indicated that the binding of myricetin in this gap region restricts the conformational transition of SLY from a monomer to an oligomer, thereby counteracting the haemolytic activity of SLY. This mechanism was verified using a haemolysis assay. These results demonstrated that myricetin is a strong candidate as a novel therapeutic agent for the treatment of Streptococcus suis infections.

TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase.

We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled O-phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λex = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions.

Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo.

Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can be easily attached, also as a mixture of two or more molecules. This non-covalent bond is stable both in cell culture and after injection into mice in vivo. Thus, we have created a highly versatile system for many applications, from the delivery of biomolecules over the targeting of cells and tissue to in vivo imaging.

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ∼20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between “deep” and “shallow” binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as “area reservoirs” leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.

Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.

Membrane Microdomain Structures of Liposomes and Their Contribution to the Cellular Uptake Efficiency into HeLa Cells.

The purpose of this study is to obtain a comprehensive relationship between membrane microdomain structures of liposomes and their cellular uptake efficiency. Model liposomes consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol (Ch) were prepared with various lipid compositions. To detect distinct membrane microdomains in the liposomes, fluorescence-quenching assays were performed at temperatures ranging from 25 to 60 °C using 1,6-diphenyl-1,3,5-hexatriene-labeled liposomes and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl. From the data analysis using the response surface method, we gained a better understanding of the conditions for forming distinct domains (Lo, Ld, and gel phase membranes) as a function of lipid composition. We further performed self-organizing maps (SOM) clustering to simplify the complicated behavior of the domain formation to obtain its essence. As a result, DPPC/DOPC/Ch liposomes in any lipid composition were integrated into five distinct clusters in terms of similarity of the domain structure. In addition, the findings from synchrotron small-angle X-ray scattering analysis offered further insight into the domain structures. As a last phase of this study, an in vitro cellular uptake study using HeLa cells was conducted using SOM clusters' liposomes with/without PEGylation. As a consequence of this study, higher cellular uptake was observed from liposomes having Ch-rich ordered domains.

G-quadruplex ligands exhibit differential G-tetrad selectivity.

A rapid and simple equilibrium-binding assay mediated by ligand-induced fluorescence quenching of fluorophore-labelled G-quadruplex (G4) structures enabled quantitative interrogation of mutually exclusive ligand binding interactions at opposed G-tetrads. This technique revealed that the ligands TmPyP4, PhenDC3, and PDS have differential chemotype-specific binding preferences for individual G-tetrads of a model genomic G4 structure.

A Systematic Method for Designing DNA Nanostructure Assembly Processes

Inspired by the complexity and diversity of biological structures that self-assemble in vivo, a fundamental goal of bionanotechnology is to self-assemble biomolecular complexes with defined architecture from a set of starting components. To design such structures, many standard methods involve constructing components that strongly bind to their neighbors through orientation-specific interactions. While these design methods ensure the target structure is favored thermodynamically, in practice the yields of the resulting assembly processes vary widely and do not conform to equilibrium predictions.To investigate why yields might be limited, we have developed a simple model of biomolecular complex self-assembly using reported rate constants and thermodynamic parameters typical for biomolecular components. Such a coarse-grained model is particularly useful in cases where we consider the assembly of engineered components and purposely design the process to include simple interactions. With these kinetic simulations, we show that even though complexes are small and equilibrium is well defined, rarely is thermodynamic equilibrium achieved over timescales commensurate with experiments. We find that rates of complex nucleation and component rearrangement together determine assembly yields.Our goal is to use this model in conjunction with experimental information to build a computer-guided, scalable design process for DNA origami components. We have used fluorescence-quenching assays to measure origami-origami reaction rates and equilibrium constants that are driven by specific Watson-Crick complementary (i.e., “sticky end”) interactions. A feedback loop between experimental measurements of a self-assembly process and a coarse-grained, computationally tractable model can guide the design of the assembly process by making it possible to focus our assembly efforts on specific interactions that make a strong difference in yields, or to redesign the process to include qualitatively new types of component interactions.