Monte Carlo Simulation of the Assembly of bis‐Biotinylated DNA and Streptavidin

We present Monte Carlo simulations of the self‐assembly of bivalent bis‐biotinylated DNA molecules with the tetravalent biotin‐binding protein streptavidin (STV). By fitting the STV binding probabilities for the four possible valencies, the modelling correctly reproduces the dependencies of various network parameters experimentally observed in an earlier study. The combined results from the experimental and theoretical studies suggest that the binding probability for divalent STV formation is about 50 times larger than for the formation of trivalent and about 200 times larger than for tetravalent STV. In accordance with the experimental results, the modelling also indicates that the mixture of an equimolar ratio of DNA and STV leads to a maximum in size of the oligomeric DNA–STV clusters formed. Furthermore, we found a percolation transition in which the DNA cluster size increases rapidly with increasing DNA concentration resulting in the formation of a single supercluster at elevated concentrations. This behaviour coincides with the occurrence of an immobile band previously observed in electrophoretic experiments, indicating the formation of extremely large DNA–STV aggregate networks.     

Accelerated Self‐Replication under Non‐Equilibrium,Periodic Energy Delivery

Self‐replication is a remarkable phenomenon in nature that has fascinated scientists for decades. In a self‐replicating system, the original units are attracted to a template, which induce their binding. In equilibrium, the energy required to disassemble the newly assembled copy from the mother template is supplied by thermal energy. The possibility of optimizing self‐replication was explored by controlling the frequency at which energy is supplied to the system. A model system inspired by a class of light‐switchable colloids was considered where light is used to control the interactions. Conditions under which self‐replication can be significantly more effective under non‐equilibrium, cyclic energy delivery than under equilibrium constant energy conditions were identified. Optimal self‐replication does not require constant energy expenditure. Instead, the proper timing at which energy is delivered to the system is an essential controllable parameter to induce high replication rates.     

Cross‐Coupling of Aryl‐Bromide and Porphyrin‐Bromide on an Au(111) Surface

Cross‐coupling is of great importance in organic synthesis. Here it is demonstrated that cross‐coupling of aryl‐bromide and porphyrin‐bromide takes place on a Au(111) surface in vacuo. The products are oligomers consisting of porphyrin moieties linked by p‐phenylene at porphyrin’s meso‐positions. The ratio of the cross‐coupled versus homocoupled bonds can be regulated by the reactant concentrations. Kinetic Monte Carlo simulations were applied to determine the activation barrier. It is expected that this reaction can be employed in other aryl‐bromide precursors for designing alternating co‐polymers incorporating porphyrin and other functional moieties.     

Determination of Allosteric Effects and Interstrand Bidentate Interactions in DNA‐Peptide Molecular Recognition

To study DNA allostery, quantitative DNase I footprinting studies were carried out on a newly designed peptide His‐Hyp‐Lys‐Lys‐(Py)₄‐Lys‐Lys‐NH₂ (HypKK‐10) containing the XHypKK (Hyp = hydroxyproline) and polyamide motifs. The interconnection of DNA footprints of peptides HypKK‐10 and the parent peptide PyPro‐12 supports the proposal that interaction network cooperativity is preferred in DNA‐peptide interactions between multiple recognition sites. A simple method of determining interstrand bidentate interactions between the peptide moieties and DNA bases is introduced. It is envisaged that interstrand bidentate interactions also participate in the relay of conformational changes to recognition sites on the complementary strands. Circular dichroism studies of the titration of peptide HypKK‐10 with an oligonucleotide duplex indicate that this peptide binds in a dimeric fashion to DNA in the minor groove. This work may prompt the design of new DNA binding ligands for the study of DNA‐peptide allosteric interactions and DNA interaction network.     

DNA‐Based Micelles: Synthesis,Micellar Properties and Size‐Dependent Cell Permeability

Functional nanomaterials based on molecular self‐assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure, and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape, and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self‐assemble into monodispersed, three‐dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine‐tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while retaining their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into the cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size‐dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity are all excellent features that make our DNA–micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems.     

A Concise Synthesis of the DNA‐Intercalating and Antimalarial Alkaloid Cryptolepine and Its Fluorescence Behaviour in Solvents of Different Polarities

A microwave‐induced rapid and facile synthesis of the DNA‐intercalating and antimalarial drug cryptolepine is described. The key step in this synthesis involves the aqueous‐phase base‐catalyzed condensation of isatin and 1‐acetyl‐1H‐indol‐3‐yl acetate which has been simplified and expedited by dielectric heating, employing an ordinary domestic microwave oven. The method transforms the synthesis of an important drug molecule from a prohibitively lengthy process to a matter of a few minutes with a much improved yield. Dual absorption and fluorescence is observed from the molecular system in solvents of different polarity thus providing valuable insight into its binding modes toward protein or DNA.     

Protein‐Specific,Multicolor and 3D STED Imaging in Cells with DNA‐Labeled Antibodies

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells.     

Dynamic and Quantitative Control of the DNA‐Mediated Growth of Gold Plasmonic Nanostructures

Reproducible and controllable growth of nanostructures with well‐defined physical and chemical properties is a longstanding problem in nanoscience. A key step to address this issue is to understand their underlying growth mechanism, which is often entangled in the complexity of growth environments and obscured by rapid reaction speeds. Herein, we demonstrate that the evolution of size, surface morphology, and the optical properties of gold plasmonic nanostructures could be quantitatively intercepted by dynamic and stoichiometric control of the DNA‐mediated growth. By combining synchrotron‐based small‐angle X‐ray scattering (SAXS) with transmission electron microscopy (TEM), we reliably obtained quantitative structural parameters for these fine nanostructures that correlate well with their optical properties as identified by UV/Vis absorption and dark‐field scattering spectroscopy. Through this comprehensive study, we report a growth mechanism for gold plasmonic nanostructures, and the first semiquantitative revelation of the remarkable interplay between their morphology and unique plasmonic properties.     

Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors,DNA‐Sensors,and Enzyme Biosensors

Impedance spectroscopy is a rapidly developing electrochemical technique for the characterization of biomaterial‐functionalized electrodes and biocatalytic transformations at electrode surfaces, and specifically for the transduction of biosensing events at electrodes or field‐effect transistor devices. The immobilization of biomaterials, e.g., enzymes, antigens/antibodies or DNA on electrodes or semiconductor surfaces alters the capacitance and interfacial electron transfer resistance of the conductive or semiconductive electrodes. Impedance spectroscopy allows analysis of interfacial changes originating from biorecognition events at electrode surfaces. Kinetics and mechanisms of electron transfer processes corresponding to biocatalytic reactions occurring at modified electrodes can be also derived from Faradaic impedance spectroscopy. Different immunosensors that use impedance measurements for the transduction of antigen‐antibody complex formation on electronic transducers were developed. Similarly, DNA biosensors using impedance measurements as readout signals were developed. Amplified detection of the analyte DNA using Faradaic impedance spectroscopy was accomplished by the coupling of functionalized liposomes or by the association of biocatalytic conjugates to the sensing interface providing biocatalyzed precipitation of an insoluble product on the electrodes. The amplified detections of viral DNA and single‐base mismatches in DNA were accomplished by similar methods. The changes of interfacial features of gate surfaces of field‐effect transistors (FET) upon the formation of antigen‐antibody complexes or assembly of protein arrays were probed by impedance measurements and specifically by transconductance measurements. Impedance spectroscopy was also applied to characterize enzyme‐based biosensors. The reconstitution of apo‐enzymes on cofactor‐functionalized electrodes and the formation of cofactor‐enzyme affinity complexes on electrodes were probed by Faradaic impedance spectroscopy. Also biocatalyzed reactions occurring on electrode surfaces were analyzed by impedance spectroscopy. The theoretical background of the different methods and their practical applications in analytical procedures were outlined in this article.     

DNA‐Modified Screen‐Printed Electrodes with Nanostructured Films of Multiwall Carbon Nanotubes,Hydroxyapatite and Montmorillonite

Nanostructured films were deposited at the surface of working electrode of the screen‐printed assembly and utilized for the surface modification with double‐stranded DNA. The basic electrochemical properties of the sensors were investigated using voltammetric methods. Modified electrodes were also characterized by scanning electron microscopy and electrochemical impedance measurements. It was found that the electrode modification with DNA and nanomodifier leads to an enhanced sensitivity of the DNA voltammetric detection. New potentialities of the utilization of the K₃[Fe(CN)₆] cyclic voltammetric signal and electrochemical impedance spectroscopy were found. The DNA‐based biosensors showed good repeability and necessary stability within several days.     

Alternating‐Current Measurements in Scanning Electrochemical Microscopy,Part 1: Principle and Theory

The development of the scanning electrochemical microscope in ac mode is presented from both experimental and theoretical point of views. The experiments are performed with the ferri/ferrocyanide redox mediator as model system. Based on analysis of the frequency‐dependent collection efficiency, diffusion between the probe and the substrate is investigated, and analysis of time constants allows evaluation of the size of the sensing area under investigation. The experimental results are in good agreement with numerical simulations.