Programmable interactions with biomimetic DNA linkers at fluid membranes and interfaces

At the heart of the structured architecture and complex dynamics of biological systems are specific and timely interactions operated by biomolecules. In many instances, biomolecular agents are spatially confined to flexible lipid membranes where, among other functions, they control cell adhesion, motility and tissue formation. Besides being central to several biological processes, \emph{multivalent interactions} mediated by reactive linkers confined to deformable substrates underpin the design of synthetic-biological platforms and advanced biomimetic materials. Here we review recent advances on the experimental study and theoretical modelling of a heterogeneous class of biomimetic systems in which synthetic linkers mediate multivalent interactions between fluid and deformable colloidal units, including lipid vesicles and emulsion droplets. Linkers are often prepared from synthetic DNA nanostructures, enabling full programmability of the thermodynamic and kinetic properties of their mutual interactions. The coupling of the statistical effects of multivalent interactions with substrate fluidity and deformability gives rise to a rich emerging phenomenology that, in the context of self-assembled soft materials, has been shown to produce exotic phase behaviour, stimuli-responsiveness, and kinetic programmability of the self-assembly process. Applications to (synthetic) biology will also be reviewed.Comment: 63 pages, revie

RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design

We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling

Membrane Scaffolds Enhance the Responsiveness and Stability of DNA-Based Sensing Circuits.

Target-induced DNA strand displacement is an excellent candidate for developing analyte-responsive DNA circuitry to be used in clinical diagnostics and synthetic biology. While most available technologies rely on DNA circuitry free to diffuse in bulk, here we explore the use of liposomes as scaffolds for DNA-based sensing nanodevices. Our proof-of-concept sensing circuit responds to the presence of a model target analyte by releasing a DNA strand, which in turn activates a fluorescent reporter. Through a combination of experiments and coarse-grained Monte Carlo simulations, we demonstrate that the presence of the membrane scaffold accelerates the process of oligonucleotide release and suppresses undesired leakage reactions, making the sensor both more responsive and robust

Scanning Probe Investigations of the Surface Self-Assembly of Organothiols and Organosilanes Using Nanoscale Lithography

Particle lithography and scanning probe lithography were applied to study the kinetics and mechanisms of surface self-assembly processes. Organothiols on Au(111) and organosilane on Si(111) were chosen as model systems for investigations at the nanoscale using atomic force microscopy (AFM). Fundamental insight of structure/property interrelationships and understanding the properties of novel materials are critical for developments with molecular devices. Methods using an AFM probe for nanofabrication have been applied successfully to prepare sophisticated molecular architectures with high reproducibility and spatial precision. The established capabilities of AFM-based nanografting were reviewed for inscribing patterns of diverse composition, to generate complicated surface designs with well-defined chemistries. Nanografting provides a versatile tool for generating nanostructures of organic and biological molecules, as well as nanoparticles. Protocols of nanografting are accomplished in liquid media, providing a mechanism for introducing new reagents for successive in situ steps for 3-D fabrication of designed nanopatterns. Because so many chemical reactions can be accomplished in solution, there are rich possibilities for chemists to design studies of other surface reactions. Surface assembly and self-polymerization of chloromethylphenyltrichlorosilane (CMPS) were investigated using test platforms of organosilanes fabricated with particle lithography. A thin film of octadecyltrichlorosilane (OTS) with well-defined nanopores was prepared on Si(111) to spatially confine the surface assembly of CMPS within nanopores of OTS. Time-dependent changes during the self-polymerization of CMPS was visualized ex situ using AFM. Molecular-level details of CMPS nanostructures were obtained from high resolution AFM images to track the growth of organosilanes on Si(111). Measurements of the heights and diameters of CMPS nanostructures provided quantitative information of the kinetics of CMPS self-polymerization. Scanning probe-based methods of nanolithography were applied to investigate the self-assembly of a tridentate organothiol, 1,1,1-tris(mercaptomethyl)heptadecane (TMMH). Multidentate adsorbates can address problems with long-term stability to oxidation observed with monothiolated n-alkylthiols. Multidentate thiol ligands demonstrate improved resistance to oxidation, thermal desorption and UV exposure. Progressive changes in surface morphology for TMMH assembly onto Au(111) was studied in situ with time-lapse AFM, monitoring changes in surface coverage at different time intervals. Nanoshaving and nanografting were used as molecular rulers to evaluate the thickness of films of TMMH

The Eighth Central European Conference "Chemistry towards Biology": snapshot

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August – 1 September 2016The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August-1 September 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting

Method and apparatus for solid state molecular analysis

The invention is a method for the formation and analysis of novel miniature deposition domains. These deposition domains are placed on a surface to form a molecular array. The molecular array is scanned with an AFM to analyze molecular recognition events and the effect of introduced agents on defined molecular interactions. This approach can be carried out in a high throughput format, allowing rapid screening of thousands of molecular species in a solid state array. The procedures described here have the added benefit of allowing the measurement of changes in molecular binding events resulting from changes in the analysis environment or introduction of additional effector molecules to the assay system. The processes described herein are extremely useful in the search for compounds such as new drugs for treatment of undesirable physiological conditions. The method and apparatus of the present invention does not require the labeling of the deposition material or the target sample and may also be used to deposit large size molecules without harming the same

Development of Diverse Size and Shape RNA Nanoparticles and Investigation of their Physicochemical Properties for Optimized Drug Delivery

RNA nanotechnology is an emerging field that holds great promise for advancing drug delivery and materials science. Recently, RNA nanoparticles have seen increased use as an in vivo delivery system. RNA was once thought to have little potential for in vivo use due to biological and thermodynamic stability issues. However, these issues have been solved by: (1) Finding of a thermodynamically stable three-way junction (3WJ) motif; (2) Chemical modifications to RNA confer enzymatic stability in vivo; and (3) the finding that RNA nanoparticles exhibit low immunogenicity in vivo. In vivo biodistribution and pharmacokinetics are affected by the physicochemical properties, such as size, shape, stability, and surface chemistry/properties, of the nanoparticles being delivered. RNA has an inherent advantage for nanoparticle construction as each of these properties can be finely tuned. The focus of this study is as follows: (1) Construction of diverse size and shape RNA nanoparticles with tunable physicochemical properties; (2) Investigation of the effect that size, shape, and nanoparticle properties have on in vivo biodistribution; (3) Development of drug encapsulation and release mechanism utilizing RNA nanotechnology; and (4) Establishment of large-scale synthesis and purification methods of RNA nanoparticles. In (1), RNA triangle, square, and pentagon shaped nanoparticles were constructed using the phi29 pRNA-3WJ as a core motif. Square nanoparticles were constructed with sizes of 5, 10, and 20 nanometers. The RNA polygons were characterized by AFM to demonstrate formation of their predicted geometry per molecular models. Furthermore, the properties of RNA polygons were tuned both thermodynamically and chemically by substitution of nucleic acid type used during nanoparticle assembly. In (2), the biodistribution of RNA nanosquares of diverse sizes and RNA polygons of diverse shapes were investigated using tumor models in nude mice. It was found that increasing the size of the nanosquares led to prolonged circulation time in vivo and higher apparent accumulation in the tumor. However, it was observed that changing of shape had little effect on biodistribution. Furthermore, the effect of the hydrophobicity on RNA nanoparticles biodistribution was examined in mouse models. It was found that incorporation of hydrophobic ligands into RNA nanoparticles causes non-specific accumulation in healthy organs, while incorporation of hydrophilic ligands does not. Lower accumulation in vital organs of hydrophobic chemicals was observed after conjugation to RNA nanoparticles, suggesting RNA has the property to solubilize hydrophobic chemicals and reduce accumulation and toxicity in vital organs. In (3), a 3D RNA nanoprism was constructed to encapsulate a small molecule fluorophore acting as a model drug. The fluorophore was held inside the nanoprism by binding to an RNA aptamer. The ability of the stable frame of the nanoprism to protect the fragile aptamer inside was evidenced by a doubling of the fluorescent half-life in a degrading environment. In (4), a method for large-scale in vitro synthesis and purification of RNA nanoparticles was devised using rolling circle transcription (RCT). A novel method for preparing circular double stranded DNA was developed, overcoming current challenges in the RCT procedure. RCT produced more than 5 times more RNA nanoparticles than traditional run-off transcription, as monitored by gel electrophoresis and fluorescence monitoring. Finally, large-scale purification methods using rate-zonal and equilibrium density gradient ultracentrifugation, as well as gel electrophoresis column, were developed

Dynamic self-assembling DNA nanosystems: design and engineering

Over the last thirty years, DNA has proven to be a great candidate for engineering nanoscale architectures. These DNA nanostructures have been applied in areas such as single-molecular analyses, nanopatterning, diagnostics and therapeutics. One of the most commonly-used techniques to engineer DNA-based two- and three-dimensional functional nanostructures is DNA origami, wherein a long single-stranded DNA (called scaffold) is folded into a predetermined shape with the help of a set of shorter oligonucleotides (called staples). This thesis discusses a brief overview of DNA nanotechnology (design, assembly and applications) and three primary projects undertaken in the area of dynamic self-assembling DNA nanosystems: 1, a self-assembly design strategy that vastly expands the utility of DNA origami, 2, a DNA origami-based reconfigurable nanosystem with potential as a force/energy balance and diagnostic tool, and 3, a collaborative initiative on computational analyses and experimental verification for improving efficiency of DNA nanoengineering