Identification of Thioflavin T Binding Modes to DNA: A Structure-Specific Molecular Probe for Lasing Applications

The binding mechanism of thioflavin T (ThT) to DNA was studied using polarized light spectroscopy and fluorescence-based techniques in solutions and in solid films. Linear dichroism measurements showed that ThT binds to DNA duplex by intercalation. Time-resolved fluorescence studies revealed a second binding mode which is the external binding to the DNA phosphate groups. Both binding modes represent the nonspecific type of interactions. The studies were complemented with the analysis of short oligonucleotides having DNA cavities. The results indicate that the interplay between three binding modes-intercalation, external binding, and binding inside DNA cavities-determines the effective fluorescence quantum yield of the dye in the DNA structures. External binding was found to be responsible for fluorescence quenching because of energy transfer between intercalated and externally bound molecules. Finally, amplified spontaneous emission (ASE) was successfully generated in the ThT-stained films and used for detecting different DNA structures. ASE measurements show that ThT-stained DNA structures can be used for designing bioderived microlasers

Transport of Live Cells under Sterile Conditions Using a Chemotactic Droplet

© 2018 The Author(s). 1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated if such droplets can be used as transporters for living cells. We developed a partially hydrophobic alginate capsule as a protective unit that can be precisely placed in a droplet and transported along chemical gradients. Once the droplets with cargo reached a defined final destination, the association of the alginate capsule and decanol droplet was disrupted and cargo deposited. Both Escherichia coli and Bacillus subtilis cells survived and proliferated after transport even though transport occurred under harsh and sterile conditions

Nonlinear absorption and nonlinear refraction: Maximizing the merit factors

Both nonlinear absorption and nonlinear refraction are effects that are potentially useful for a plethora of applications in photonics, nanophotonics and biophotonics. Despite substantial attention given to these phenomena by researchers studying the merits of disparate systems such as organic materials, hybrid materials, metal-containing molecules and nanostructures, it is virtually impossible to compare the results obtained on different materials when varying parameters of the light beams and different techniques are employed. We have attempted to address the problem by studying the properties of various systems in a systematic way, within a wide range of wavelengths, and including the regions of onephoton, two-photon and three-photon absorption. The objects of our studies have been typical nonlinear chromophores, such as π-conjugated molecules, oligomers and polymers, organometallics and coordination complexes containing transition metals, organometallic dendrimers, small metal-containing clusters, and nanoparticles of various kinds, including semiconductor quantum dots, plasmonic particles and rare-earth doped nanocrystals. We discuss herein procedures to quantify the nonlinear response of all of these systems, by defining and comparing the merit factors relevant for various applications

Multiphoton absorption in amyloid protein fibres

Fibrillization of peptides leads to the formation of amyloid fibres, which, when in large aggregates, are responsible for diseases such as Alzheimer's and Parkinson's. Here, we show that amyloids have strong nonlinear optical absorption, which is not present in native non-fibrillized protein. Z-scan and pump-probe experiments indicate that insulin and lysozyme β-amyloids, as well as α-synuclein fibres, exhibit either two-photon, three-photon or higher multiphoton absorption processes, depending on the wavelength of light. We propose that the enhanced multiphoton absorption is due to a cooperative mechanism involving through-space dipolar coupling between excited states of aromatic amino acids densely packed in the fibrous structures. This finding will provide the opportunity to develop nonlinear optical techniques to detect and study amyloid structures and also suggests that new protein-based materials with sizable multiphoton absorption could be designed for specific applications in nanotechnology, photonics and optoelectronics

Coupled Growth and Division of Model Protocell Membranes

The generation of synthetic forms of cellular life requires solutions to the problem of how biological processes such as cyclic growth and division could emerge from purely physical and chemical systems. Small unilamellar fatty acid vesicles grow when fed with fatty acid micelles and can be forced to divide by extrusion, but this artificial division process results in significant loss of protocell contents during each division cycle. Here we describe a simple and efficient pathway for model protocell membrane growth and division. The growth of large multilamellar fatty acid vesicles fed with fatty acid micelles, in a solution where solute permeation across the membranes is slow, results in the transformation of initially spherical vesicles into long thread-like vesicles, a process driven by the transient imbalance between surface area and volume growth. Modest shear forces are then sufficient to cause the thread-like vesicles to divide into multiple daughter vesicles without loss of internal contents. In an environment of gentle shear, protocell growth and division are thus coupled processes. We show that model protocells can proceed through multiple cycles of reproduction. Encapsulated RNA molecules, representing a primitive genome, are distributed to the daughter vesicles. Our observations bring us closer to the laboratory synthesis of a complete protocell consisting of a self-replicating genome and a self-replicating membrane compartment. In addition, the robustness and simplicity of this pathway suggests that similar processes might have occurred under the prebiotic conditions of the early Earth.Exobiology Program (U.S.) (Grant EXB02- 0031-0018)United States. National Aeronautics and Space Administration (Exobiology Program) (Grant EXB02-0031-0018)Howard Hughes Medical Institute (Investigator

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell

Promotion of protocell self-assembly from mixed amphiphiles at the origin of life

Vesicles formed from single-chain amphiphiles (SCAs) such as fatty acids probably played an important role in the origin of life. A major criticism of the hypothesis that life arose in an early ocean hydrothermal environment is that hot temperatures, large pH gradients, high salinity and abundant divalent cations should preclude vesicle formation. However, these arguments are based on model vesicles using 1–3 SCAs, even though Fischer–Tropsch-type synthesis under hydrothermal conditions produces a wide array of fatty acids and 1-alkanols, including abundant C10–C15 compounds. Here, we show that mixtures of these C10–C15 SCAs form vesicles in aqueous solutions between pH ~6.5 and >12 at modern seawater concentrations of NaCl, Mg2+ and Ca2+. Adding C10 isoprenoids improves vesicle stability even further. Vesicles form most readily at temperatures of ~70 °C and require salinity and strongly alkaline conditions to self-assemble. Thus, alkaline hydrothermal conditions not only permit protocell formation at the origin of life but actively favour it

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

Background: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 59 or 29 OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. Methodology/Principal Findings: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 59 phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2.A1.A3.A4.A0.A6.A8.A10 and T2.T3.T4.T6<T1.T8.T10. Bridging T’s generally gave higher yield of ligated product than bridging A’s. ATP concentrations above 33 mM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (.90%) joined a correctly paired, or T:G wobble-paired, substrate on the 39 side of the ligation junction while discriminating approximately 100-fold against most mispaire

Multilevel Selection in Models of Prebiotic Evolution II: A Direct Comparison of Compartmentalization and Spatial Self-Organization

Multilevel selection has been indicated as an essential factor for the evolution of complexity in interacting RNA-like replicator systems. There are two types of multilevel selection mechanisms: implicit and explicit. For implicit multilevel selection, spatial self-organization of replicator populations has been suggested, which leads to higher level selection among emergent mesoscopic spatial patterns (traveling waves). For explicit multilevel selection, compartmentalization of replicators by vesicles has been suggested, which leads to higher level evolutionary dynamics among explicitly imposed mesoscopic entities (protocells). Historically, these mechanisms have been given separate consideration for the interests on its own. Here, we make a direct comparison between spatial self-organization and compartmentalization in simulated RNA-like replicator systems. Firstly, we show that both mechanisms achieve the macroscopic stability of a replicator system through the evolutionary dynamics on mesoscopic entities that counteract that of microscopic entities. Secondly, we show that a striking difference exists between the two mechanisms regarding their possible influence on the long-term evolutionary dynamics, which happens under an emergent trade-off situation arising from the multilevel selection. The difference is explained in terms of the difference in the stability between self-organized mesoscopic entities and externally imposed mesoscopic entities. Thirdly, we show that a sharp transition happens in the long-term evolutionary dynamics of the compartmentalized system as a function of replicator mutation rate. Fourthly, the results imply that spatial self-organization can allow the evolution of stable folding in parasitic replicators without any specific functionality in the folding itself. Finally, the results are discussed in relation to the experimental synthesis of chemical Darwinian systems and to the multilevel selection theory of evolutionary biology in general. To conclude, novel evolutionary directions can emerge through interactions between the evolutionary dynamics on multiple levels of organization. Different multilevel selection mechanisms can produce a difference in the long-term evolutionary trend of identical microscopic entities