Recent patents in bionanotechnologies: nanolithography, bionanocomposites, cell-based computing and entropy production

Recent Patents on Nanotechnology, 2: pp. 1-7This article reviews recent disclosures of bio-inspired, bio-mimicked and bionanotechnologies. Among the patents discussed is a nanoscale porous structure for use in nanocomposites and nanoscale processing. Patents disclosing methods for printing biological materials using nanolithography techniques such as dip-pen technology are discussed, as are patents for optimizing drug design. The relevance of these technologies to disease prevention, disease treatment and disease resistance is discussed. The paper closes with a review of cell-based computing and a brief examination of how information technology has enabled the development of these technologies. Finally a forecast of the how these technologies are likely to accelerate global entropization is discussed as well as a new classification of machine types

Advances in High-Speed Atomic Force Microscopy

High-speed atomic force microscopy (HS-AFM) is a scanning probe technique capable of recording processes at the nanometre scale in real time. By sequentially increasing the speed of individual microscope components, images of surfaces can be recorded at up to several images per second. We present a HS-AFM platform composed of custom¿built measurement head, controller and software, scanners and amplifiers that is shared with the community in an open¿hardware fashion. A new scanner design combined with an advanced control system is shown. The simple addition of a secondary actuator to widely available tube scanners increases the scan speed by over an order of magnitude while allowing for a 130 ¿m × 130 ¿m wide field of view, which is not possible with traditional high¿speed scanner designs. Controllers beyond standard proportional-integral controllers are capable of significantly increasing imaging speed by anticipating resonances. Such filters are cumbersome to design with conventional methods. It is shown how convex optimization can be used to design optimal controllers with guaranteed stability for atomic force microscopy in an automated fashion. By integrating two lasers into the small spot¿size optics of an AFM readout head we are able to use the first laser for detecting the deflection of the smallest, and thus fastest currently available high¿speed cantilevers, while using the second for photo¿thermal actuation. Using this instrument, we demonstrate multi¿frequency atomic force microscopy (MF-AFM) at previously not accessible frequencies of more than 20 MHz. By employing the driving laser not for resonant excitation as is usual in dynamic AFM, a new imaging mode, photothermal off-resonance tapping (PORT) is presented. By repeatedly thermally bending the cantilever below it¿s resonant frequency, the surface is probed at a rapid rate. The resulting force is extracted from the deflection of the cantilever in time¿ domain at real time and used for feedback and image generation. The dynamic and static force contributions in both PORT and state of the art high-speed amplitude modulation atomic force microscopy (AM-AFM) are measured and analyzed in detail. It is shown that by decoupling the driving frequency from the resonant frequency the dynamic tip¿sample impact forces can be drastically reduced when compared to resonance based AFM modes. SAS-6 is a centriolar scaffolding protein with a crucial role in the duplication of centrioles, which are the main microtubule organizing organelle of eukaryotic cells. Defects in centriole duplication are associated with cancer and microencephaly. To understand these defects, is therefore important to understand the kinetics of SAS-6. In¿vitro, SAS-6 polymerizes into rings of between eight and ten monomers. Using the new PORT mode we are able to study the dynamic assembly of SAS-6. It is shown how SAS-6 rings can not only assemble by canonical one-by-one addition, but can form as a fusion of larger, already assembled fragments. Finally, it is shown how PORT can be used to observe fast processes of and on living cells. The adhesion and detachment of thrombocyte cells is studied. Membrane disruptive effects are shown on gram¿negative as well as gram¿positive bacteria

DNA ORIGAMI ACTUATION AS A POWERFUL DYNAMIC AND TUNABLE ARCHITECTURE FOR PLASMONIC STRUCTURE

2013/2014In questa tesi presento il mio lavoro di ricerca di Dottorato in Nanotecnologie. Questo studio è concentrato sull'uso di nanotecnologia a DNA come strumento per la creazione di strutture nano-biologiche e funzionalizzazione di particelle d'oro. Le nano-particelle d'oro sono state largamente studiate e le loro proprietà sono state sfruttate per importanti applicazioni come la spettroscopia Raman, la rilevazione biologica e la terapia medica. Queste nano-particelle sono caratterizzate da una risonanza ottica plasmonica e proprietà di dispersione della luce ben definite ed esistono numerosi protocolli di provata efficienza per la loro funzionalizzazione chimica. Tra questi, il protocollo di auto-assemblaggio di DNA si è dimostrato eccellente nel comporre strutture di nano-particelle con dimensioni e forma controllate. Questo approccio è stato impiegato per l'ingegnerizzazione di proprietà ottiche, per la creazione di "hot spot” nel campo plasmonico in aggregati di nano-particelle e anche per la formazione di righelli plasmonici con dimeri di nano-particelle nei quali la loro spaziatura è controllata con precisione nanometrica. In questo studio confronto due strategie per la formazione di dimeri di nano-particelle d'oro usando l'ibridizzazione del DNA. Una di queste strategie mi ha permesso di raggiungere una al resa del 26% di formazioni di dimeri rispetto al totale delle AuNP, senza ulteriori procedure di filtrazione, dato che rappresenta il valore più alto riportato in letteratura; inoltre questo dato è stato replicato utilizzando sequenze di DNA molto corte, fino ad 11 nucleotidi, condizione che normalmente riduce l’efficienza del processo. Nella seconda parte della mia tesi, ho combinato le proprietà plasmoniche delle nano-particelle d'oro con strutture a DNA origami in modo da creare sistemi ibridi tra di loro. Questa combinazione mi ha permesso di esplorare architetture innovative per la il controllo della plasmonica con la prospettiva di essere un punto di partenza per lo sviluppo di biosensori. Ho sviluppato una strategia per un controllo innovativo, reversibile e continuo della risonanza plasmonica usando un'attuazione basata su DNA origami. Il meccanismo di attuazione è basato sull'ibridizzazione del DNA, in particolare si è visto uno spostamento del picco di risonanza fino a 6 nm utilizzando tre sequenza di DNA diverse. Il sistema proposto è potrà essere utilizzato per lo studio dei meccanismi di ibridazione di DNA in condizioni di stress controllato, oppure potrà essere usato come piattaforma per un controllo continuo della posizione della risonanza plasmonica o in spettroscopia Raman.XXVII Ciclo198

Multiplexed single molecule observation and manipulation of engineered biomolecules

Molecular processes in organisms are often enabled by structural elements resilient to mechanical forces. For instance, the microbial and hierarchical cellulosome protein system comprises enzymes and the receptor-ligand complexes Cohesin-Dockerin (Coh-Doc), that act in concert for the efficient hydrolysis of plant polysaccharides. The Coh-Doc complexes can withstand remarkably high forces to keep host cells and enzymes bound to their substrates in the extreme environmental conditions the microorganisms frequently live in. This work focuses on the investigation of mechanical stability of such biomolecules on the single-molecule level. The highly symmetric binding interface of the Coh-Doc type I complex from Clostridium thermocellum, enables two different binding conformations withcomparable affinity and similar strength. I was able to show that both conformations exist in the wild-type molecules and are occupied under native conditions. I further characterized one of the strongest non-covalent protein complexes known, Coh-Doc type III from Ruminococcus flavefaciens by elucidating the pivotal role of the adjacent xModule domain for the mechanical stabilization of the whole complex and the role of the bimodal rupture force distribution. Such large forces impair accuracy of measured contour length increments in unfolding studies by inducing conformational changes in poly-ethylene glycol (PEG) linkers in aqueous buffer systems. This problemwas solved by introducing elastin-like polypeptides (ELP) as surface tethers. Having a peptide backbone similar to that of unfolded proteins, ELP linkers do not alter accuracy of the single-molecule force spectroscopy (SMFS) assay. To provide high throughput and precise comparability, I worked on a microfluidic platform for the in vitro protein synthesis and immobilization. The Coh-Doc system was hereby integrated as a binding handle for multiplexed measurements of mechanostability. Employing a single AFM probe to measure multiple different molecules facilitates force precision required to shed light onto molecular mechanisms down to the level of single amino acids. I also applied the Coh-Doc complex to a purely protein based single-molecule cut and paste assay for the bottom-up assembly of molecular systems for quick phenotyping of spatial arrangements. With this system, interactions in enzymatic synergies can be studied by defined positioning patterns on the single molecule level. To understand and design force responses of complex systems, I complemented the investigation of protein systems with SMFS studies on DNA Origami structures. The results of SMFS on DNA were compared to a simulation framework. Despite their difference in force loading rates, both methods agree well within their results, enabling better fundamental understanding of complex molecular superstructures.Molekulare Prozesse in Organismenwerden oft von Strukturelementen ermöglicht, die mechanischen Kräften standhalten können. Ein Beispiel hierfür ist das mikrobielle und hierarchisch aufgebaute Proteinsystem des Zellulosoms. Enzyme und die Rezeptor-Liganden Komplexe Cohesin-Dockerin (Coh-Doc) arbeiten hierbei für die effiziente Hydrolyse von pflanzlichen Polysacchariden zusammen. Die Coh-Doc Komplexe können bemerkenswerten Kräften standhalten, um in den extremen Umweltbedingungen, in denen die Mikroorganismen teilweise leben, die Wirtszellen und Enzyme an ihre Substrate binden zu können. Die vorliegende Arbeit untersucht den Einfluss von mechanischer Kraft auf solche Biomoleküle mittels Einzelmolekülmessungen. Die hohe Symmetrie des Bindeinterfaces des Coh-Doc Typ I Komplexes aus Clostridium thermocellum ermöglicht zwei verschiedene Konformationen, die vergleichbare Affinität und Stärke aufweisen. Im Rahmen dieser Arbeit konnte ich beide in denWildtyp-Molekülen und unter nativen Bedingungen nachweisen. Eines der stärksten bekannten nicht-kovalenten Rezeptor-Liganden Systeme, Coh- Doc Typ III aus Ruminococcus flavefaciens wurde charakterisiert, und die Kernrolle des benachbarten xModuls für die Stabilität des gesamten Komplexes sowie die Rolle der bimodalen Kraftverteilung untersucht. Solch hohe Kräfte vermindern die Genauigkeit der gemessenenKonturlängeninkremente von Proteinentfaltungen, indem sie Konformationsänderungen der Poly- Ethylenglykol (PEG) Oberflächenanker in wässrigen Puffersystemen verursachen. Mit Elastin-ähnlichen Polypeptiden (ELP) als Anker wurde dieses Problem gelöst: durch die Ähnlichkeit des Peptid-Rückgrates von ELPs mit dem entfaltener Proteine beeinflussen diese die Genauigkeit des Experiments nicht. Für die Optimierung von Messdurchsatz und Vergleichbarkeit entwickelte ich an einer Mikrofluidik-Plattform zur in vitro Proteinsynthese und -immobilisierung. Das Coh-Doc System wurde hierbei als Binde-Molekül für gemultiplexte Messungen integriert. Die dadurch ermöglichte Nutzung einer einzigen AFM Messsonde für die Messung verschiedener Moleküle erlaubt die nötige Kraftpräzision, um molekulare Mechanismen bis auf die Ebene einzelner Aminosäuren aufzuklären. Des weiteren habe ich den Coh-Doc Komplex in einem rein auf Proteininteraktionen basierten ’Cut and Paste’ Assay für den modularen Aufbau molekularer Systeme implementiert. Dieses ermöglicht schnelle Phänotypisierung geometrischer Anordnunungen und die Untersuchung von Wechselwirkung zwischen Enzymen mittels definierter Positionierung auf Einzelmolekülebene. Um die Kraftantwort komplexer Systeme besser verstehen und letztendlich gestalten zu können, ergänzte ich die Untersuchung von Proteinsystemen mit derer von DNA-Origami Strukturen. Die Ergebnisse der Kraftspektroskopie an DNA wurden mit Computersimulationen verglichen, und trotz des großen Unterschieds ihrer Ladungsraten stimmen beide Methoden gut überein. Dadruch legen sie die Grundlagen für ein besseres Verständnis komplexer molekularer Superstrukturen

Konnektivität molekularer Domänen bei der kraftinduzierten Entfaltung einzelner Biomoleküle

In den zellulären Stoffwechsel- und Signalnetzwerken existiert eine Vielzahl von logischen Abhängigkeiten, die auf Prozesse auf molekularer Ebene zurückzuführen sind. So lässt sich beispielsweise die Effizienz einer biochemischen Reaktion über Enzyme regulieren, deren Aktivitätsgrad von äußeren Parametern abhängt. Kraft stellt eine dieser Einflussgrößen dar. Diese Arbeit befasst sich damit, das Verhalten mehrerer, logisch verknüpfter, molekularer Domänen unter Krafteinwirkung zu studieren und sich deren Eigenschaften für nanotechnologische Verfahren zu Nutze zu machen. Neben der Untersuchung von in der Natur vorkommenden Proteinen mit multiplen Domänen wurden artifizielle DNA- und proteinbasierte Systeme mit verschiedener Bindungsstärke konstruiert. Dies ermöglicht den gerichteten Transport einzelner, molekularer Bausteine mit der Präzision eines Rasterkraftmikroskopes im Nanometer-Bereich. Mithilfe dieser Single-Molecule Cut-and-Paste (SMCP) Technik können auf der Basis gerichteter, molekularer Erkennung räumliche Arrangements funktioneller Bausteine geschaffen werden. Diese lassen sich mittels Fluoreszenzmikroskopie als isoliertes System betrachten. Die Zielsetzung bei der Untersuchung der natürlichen Systeme war es, deren Abhängigkeiten zu verstehen und herauszufinden, wie sich diese mit ihrer Funktion und den an das Protein gestellten Umgebungsbedingungen in Einklang bringen lassen. Die dabei gewonnene Erkenntnis liefert nicht nur wichtige Beiträge zur biologischen und medizinischen Grundlagenforschung, sondern kann, wie am Beispiel der SMCP-Technik ersichtlich, auch hilfreich bei der Entwicklung neuartiger Messmethoden der molekularen Bio- und Nanotechnologie sein. Mittels Einzelmolekülkraftspektroskopie im „Konstante-Kraft“ (engl. Force-Clamp) Modus wurde die Kooperativität der fünf Proteindomänen des Enzyms Titinkinase untersucht. Dieses Muskelprotein wandelt in der Skelett- und Herzmuskulatur mechanische in biochemische Signale um und regelt dadurch den Umsatz weiterer Proteine und die Expression von Genen. Es wird gezeigt, dass sich die einzelnen mechanisch induzierten Entfaltungsschritte gegenseitig bedingen und dass dies inhärent durch die molekulare Faltung des Proteins vorgegeben wird. Da Kraft zum natürlichen Parameterraum dieses Moleküls gehört, muss seine Struktur an kraftinduzierte konformationelle Änderungen angepasst sein. Durch die Abhängigkeit der Energiebarrieren während der Entfaltung wird gewährleistet, dass stabilisierende und enzymatisch wirksame Domänen nicht vor regulatorischen Domänen entfalten. Myosin-Light-Chain Kinase (MLCK) ist ein weiteres Muskelenzym, bei dem es Hinweise auf eine mechanische Aktivierbarkeit gibt. Einzelmolekülexperimente dieser Dissertation zeigen, dass die Entfaltung der Kinase ebenfalls in mehreren Schritten vonstatten geht und dass einer der Zwischenzustände durch ATP-Bindung stabilisiert wird. Die absoluten Entfaltungskräfte liegen dabei unter denen der Titinkinase, was der Hypothese der mechanischen Aktivierbarkeit entgegenkommt. Als weiteres System wurde das Cellulosom des thermophilen Bakteriums Clostridium Thermocellum auf seine mechanische Stabilität überprüft. Cellulosome sind an der Außenseite von Bakterien und Pilzen verankerte Proteinkomplexe, die in der Lage sind Lignozellulose zu zersetzen. Bei der Prozessierung der Cellulose können im Cellulosom hohe Scherkräfte auftreten, da dieses das gesamte Bakterium mit dem makromolekularen Substrat verknüpft. Mittels AFM-basierter Kraftspektroskopie wurde die Wirkung von Kraft auf einen Verbund verschiedener Konstituenten eines Cellulosoms untersucht. Es wird gezeigt, dass sich der Komplex im Vergleich zu anderen Biomolekülen durch eine extrem hohe mechanische Stabilität auszeichnet. Innerhalb der hohen Entfaltungskräfte besteht eine Hierarchie für die verschiedenen Komponenten. Bei vergleichsweise niedrigen Kräften entfalten die enzymatischen Domänen gefolgt von mittleren Kräften für das Entkoppeln der Enzyme mit dem Bindungspartner Cohesin. Sehr hohen Kräften halten die intramolekularen Wechselwirkungen der Cohesine und der Cellulose bindenden Domänen stand. Die Abstufung hoher Stabilitäten stellt eine sehr gute Anpassung an die natürlichen Anforderungen des Proteinkomplexes dar. Für die durchgeführten Messungen wurde ein modulares Kraftmikroskop (AFM) entwickelt, das sich mit einem einzelmolekülsensitiven Fluoreszenzmikroskop kombinieren lässt. Die spezielle Konstruktion weist eine extrem hohe mechanische Stabilität auf. Mittels einer photothermischen Regelung kann das AFM darüber hinaus für sensitive Bildgebung weicher molekularer Oberflächen oder in einen extrem schnellen kraftspektroskopischen Messmodus mit konstanter Zugkraft verwendet werden. Die akkurate Arbeitsweise des Systems wurde in einem internationalen Vergleichsversuch bestätigt

Programmable and Multifunctional DNA-Based Materials for Biomedical Applications

DNA encodes the genetic information; recently, it has also become a key player in material science. Given the specific Watson–Crick base‐pairing interactions between only four types of nucleotides, well‐designed DNA self‐assembly can be programmable and predictable. Stem‐loops, sticky ends, Holliday junctions, DNA tiles, and lattices are typical motifs for forming DNA‐based structures. The oligonucleotides experience thermal annealing in a near‐neutral buffer containing a divalent cation (usually Mg2+) to produce a variety of DNA nanostructures. These structures not only show beautiful landscape, but can also be endowed with multifaceted functionalities. This Review begins with the fundamental characterization and evolutionary trajectory of DNA‐based artificial structures, but concentrates on their biomedical applications. The coverage spans from controlled drug delivery to high therapeutic profile and accurate diagnosis. A variety of DNA‐based materials, including aptamers, hydrogels, origamis, and tetrahedrons, are widely utilized in different biomedical fields. In addition, to achieve better performance and functionality, material hybridization is widely witnessed, and DNA nanostructure modification is also discussed. Although there are impressive advances and high expectations, the development of DNA‐based structures/technologies is still hindered by several commonly recognized challenges, such as nuclease instability, lack of pharmacokinetics data, and relatively high synthesis cost. </p

Understanding the Molecular Mechanism of Single-Strand Annealing Homologous DNA Recombination in Viruses, by Cryo-Electron Microscopy

The single-strand annealing homologous recombination (SSA) is one of the dsDNA break repair pathways, and albeit its importance from bacteria to bacteriophages, its molecular function is still unknown. The SSA reaction is catalysed by the enzyme complexes known as Exonuclease Annealase Two-component Recombinase (EATRs). The RecT and ORF6 proteins are single-stranded DNA-binding and annealing proteins expressed in E. coli and Kaposi’s sarcoma-associated herpesvirus (KSHV), respectively. RecT has already been shown to catalyse the SSA reaction. Although ORF6 has been shown to bind to ssDNA, further experimental evidence is needed to solidify its annealase activity. Since structure can dictate the function, this thesis aimed to determine the structure of the annealases RecT and ORF6 using a state-in-art cryo-electron microscopy technique. Furthermore, the shadow-casting EM technique has been established by optimising it for the equipment available at UOW, which is helpful for imaging the substrate DNA intermediates and the nucleoprotein complexes formed during SSA to better understand the molecular mechanistic details of this reaction. This thesis includes the details about RecT and ORF6 proteins’ cloning, expression, and purification, which were further optimised for purity and homogeneity for cryo-electron microscopy with the help of negative staining electron microscopy (NSEM). Additionally, based on several NSEM analyses, the C-terminal His-tag containing RecT (RecTCH) oligomerisation on ssDNA was studied, and a general mechanism of its oligomerisation is described. Unfortunately, during the RecTCH protein’s cryo-EM sample optimisation, the LiRecT structure was published by another group. Therefore, work on that project was ceased at that point. Several novel findings on ORF6 are reported in this thesis. Primarily, the concentration of the purified protein was increased 3 times more than the reports in the literature. Based on the NSEM and preliminary cryo-EM map of ORF6, it is shown that the ORF6 structure overall resembles the HSV1-ICP8 protein. Further, based on the steady-state and time-resolved fluorescence resonance energy transfer (FRET) experiments, a model for the ORF6 annealing mechanism is suggested. Towards generating a high-resolution structure, ORF6 monomers and filaments were optimised and imaged by using cryo-EM. Processing a data set obtained from a monomeric ORF6 sample showed the presence of conformational heterogeneity in the particles, which was expected as the ORF6 AlphaFold model shows that the N-terminal and C-terminal domains are connected by an 18 amino acids long loop, allowing C-terminal domain to be relatively flexible to move around. Processing of another data set obtained from a sample containing ORF6 filaments generated 2-dimensional averages that look promising for generating a high-resolution structure. This thesis also shows the details related to the installation and optimisation of the shadowing technique using a modern material, graphene oxide (GO), as a support film. This technique involves optimising both sample preparation and instrumentation for metal evaporation and deposition. For sample preparation, GO was deposited on cryo-EM holey grids, on which the sample was mounted. For instrumentation optimisation, a DENTON brand evaporator was used. The grid stage was re-engineered using AutoCAD to achieve the finest metal evaporation, and parameters such as amperage, vacuum, metal thickness, and angles were optimised. The optimised parameters were used to shadow-cast different lengths of DNA and their complexes with proteins, and good contrast images were acquired for qualitative and quantitative analyses. Overall, this thesis presents two main novel findings. First, RecTCH monomers oligomerise into an open ring-shaped structure, which stacks together to generate short filaments. Second, to anneal two complementary ssDNA strands, ORF6 first forms filaments with both ssDNA, which then come in contact with each other rapidly to anneal the complementary strands. Once the annealing finishes, the annealed dsDNA is released from the filaments as the filaments fall apart into monomers. We also found that ORF6 monomers oligomerise to form the helical and non-helical filaments in the presence of DTT+Mg2+ and DTT-containing buffer, respectively