Tight Bounds for Active Self-Assembly Using an Insertion Primitive

We prove two tight bounds on the behavior of a model of self-assembling particles introduced by Dabby and Chen (SODA 2013), called insertion systems, where monomers insert themselves into the middle of a growing linear polymer. First, we prove that the expressive power of these systems is equal to context-free grammars, answering a question posed by Dabby and Chen. Second, we prove that systems of $k$ monomer types can deterministically construct polymers of length $n = 2^{\Theta(k^{3/2})}$ in $O(\log^{5/3}(n))$ expected time, and that this is optimal in both the number of monomer types and expected time.Comment: To appear in Algorithmica. An abstract (12-page) version of this paper appeared in the proceedings of ESA 201

ENGINEERING DNA-BASED NANOCHANNELS AND VESICLES FOR CONTROLLED MOLECULAR TRANSPORT

For the past two decades, synthetic transmembrane nanopores and nanochannels have become powerful tools in biosensing and single-molecule studies. Due to the ease of rational design and advancements in DNA functionalization, DNA has been established to be versatile building blocks for the bottom-up fabrication of nanostructures. Recently, DNA-based nanopores in both small diameters (1-2 nm) showing transport of ions and small molecules and large diameters (5-10 nm) showing transport of proteins across lipid bilayer membranes were reported. Nevertheless, those DNA nanopores have lengths below 100 nm, and the molecular transport only occurs across lipid membranes. It remains unknown if longer nanochannels can be constructed for transport over extended distances. Such nanochannels of longer lengths can be potentially used as conduits for carrying molecules on the cell-size scale or between compartments apart. We have designed a microns-long DNA nanochannel 7 nm inner diameter that inserts onto the lipid membranes of giant unilamellar vesicles and allows the transport of small molecules through its barrel. Kinetics analysis suggests a continuum diffusion model can describe the transport phenomenon within the DNA nanochannel. The reduced transport upon bindings of DNA origami caps to the channel ends reveals the molecules mainly transport from one channel end to the other rather than leak across channel walls. We further design a DNA nanopore-cap system that responds to specific DNA sequences. In combination with giant unilamellar vesicles that encapsulate glucose molecules, we present a biosensor system consisting of capped DNA nanopores and vesicles that can detect and amplify nanomolar DNA signals millimolar glucose outputs. The DNA-based biosensor we developed shows the potentials to be used as point-of-care nucleic acid diagnostic devices. Another challenge in using DNA nanochannels or other DNA-based nanostructures in biological environments or cell culture is that they may be degraded by enzymes found in these environments, such as nucleases. To improve the DNA nanostructures' stability, we demonstrate a means by which degradation can be reversed in situ through the repair of nanostructure defects. The ability to repair nanostructures, such as DNA nanochannels, could allow particular structures or devices to operate for long periods of time and might offer a single means to resist different types of chemical degradation

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

Reconfigurable DNA-nanochambers as dynamic compartmentalization systems

Dynamische DNA-Nanotechnologie repräsentiert eine innovative Methodik biomimeti-sche Nanostrukturen mit zunehmender Komplexität und Präzision aufzubauen. Ein großer Vorteil bei der Verwendung dieser Technik liegt in der gesamten räumlich und zeitlich kontrollierbaren Steuerung der Systeme im Nanometerbereich. Die Konfigu-rierbarkeit der Strukturen wurde in dieser Arbeit über sogenannte DNA Haarnadel-motive gesteuert. Mit Hilfe von temperaturabhängiger Förster-Resonanzenergietransfer (FRET) Spektroskopie war es somit möglich, die mechani-sche Kapazität und die Energie integrierter Ensembles dieser DNA Motoren innerhalb eines DNA Origami Systems zu bestimmen. Das Ergebnis ist ein neuartiges Modell, welches die Energielandschaft der Haarnadelmotive beschreibt. Dafür wurde das Nearest-Neighbor Modell, welches die thermodynamische Energie des DNA-Duplexes in der offenen Haarnadelform beschreibt, mit der freien entropischen Ener-gie der Einzelstrang DNA (geschlossene Form), die mittels des Worm-like Chain Algorithmus bestimmt wurde, miteinander kombiniert. Das gewonnene Verständnis über die Steuerung und Manipulation molekularer Kräfte ist essentiell und fundamen-tal für die Entwicklung und Konstruktion anspruchsvollerer Nanomaschinen und ge-währt zudem Einblick in die Funktionsweise komplexer molekularer Prozesse. Weiterhin war es möglich, durch die Verwendung dieser Methodik, strategisch zwei spezifische DNA Aptamere (TBA1 und TBA2) innerhalb eines DNA-Origami Rahmens zu integrieren, welches die Einkapselung der Serin Protease Thrombin ermöglichte. Die entwickelte Nanofabrik erlaubte somit die 1:1 host-guest Komplexierung ohne die natürlichen Eigenschaften des Proteins zu verändern, welches vergleichbar mit na-türlichen Kompartiment Systemen ist. Die Ergebnisse der Analysen zeigten, dass die Bindungsaffinität der Aptamer Liganden zum Protein innerhalb des Origami Systems und die katalytische Aktivität von Thrombin stark erhöht werden konnten und dass die geometrische Integration der Liganden eine effektive Methodik für die selektive Komplexierung und Manipulation eines gewünschten und vorher ausgewählten Pro-teins darstellt. Zusammenfassend konnte in dieser Arbeit das hohe Potential der DNA Nanotechnologie für die Konstruktion programmierbarer, bioinspirierter und künstlicher Nanokompartimentsysteme bewiesen werden, die für die Speicherung und den Transport spezifischer Materialien/Proteine zu definierten Zielorten innerhalb der Zelle verwendet werden können.Dynamic DNA nanotechnology offers an innovative opportunity to build up biomimetic nanostructures with increasing complexity and precision, depending on the overall spatial and temporal control of matter distribution with nanometer accuracy and in a trigger dependent manner. Mechanically switchable hairpin motifs thereby offer the possibility to perform DNA-induced conformational transitions. By means of tempera-ture dependent FRET spectroscopy it was possible to explore the operational capa-bilities, energetics and mechanical performance of a distinct collective ensemble of hairpin motifs tethered to a large DNA origami framework with the result of a novel hybrid spring model to describe the energy landscape of the integrated switchable hairpins. Consequently, the thermodynamic nearest-neighbor energy of the duplex DNA with the entropic free energy of single-stranded DNA estimated using a worm-like chain approximation was combined. Understanding of how mechanical forces can be gathered and manipulated at the molecular level is fundamental for the de-velopment of more sophisticated nanodevices and may help to gain more insights into the performance of complex natural molecular machines. Additionally, the strategic positioning of two G4-motifs (TBA1 and TBA2) within the inner cavity of the DNA frame demonstrated the possibility to form a 1:1 host-guest complex, without altering the natural properties of the encapsulated protein, thus emulating some of the fundamental properties of natural compartmentalization sys-tems. The results demonstrated that the binding affinity and activity of the thrombin were greatly enhanced by caging it within the origami frame and that defined geo-metric arrangements of the internalized aptamer ligands can be used to develop a tool for selective encapsulation and manipulation of desired molecular cargos. In conclusion, this work shows the high potential of DNA nanotechnology to build up programmable, dynamical, bioinspired artificial nanovessels, which might be used for the storage and delivery of materials and desired protein targets at precise cellular locations

DNA-Based Mimics of Membrane Proteins Lipid-DNA Interactions Determine Function

Nucleic acids, particularly DNA, are used as a nanoscale building material, due to their unique controllability via complementarity of base pairing. One of the potential applications of DNA nanotechnology is creating synthetic constructs mimicking function of membrane proteins. These natural molecular machines function embedded in the lipid bilayer. Similar membrane attachment of DNA-based structures is achieved by modifying the nucleic acid with hydrophobic anchors, most commonly cholesterol. Aiming at developing a fully functional and controllable synthetic membrane construct, the first step I undertook was to understand and utilize fundamental interactions between molecules: DNA, cholesterol and lipids. Instead of starting with a complicated DNA-based model mimicking protein architecture, here I have created a set of simple systems that allowed me to examine the major interactions between involved molecules. This work describes four aspects of the DNA-lipid systems that I have built and studied experimentally. Firstly, I have analysed the effects of membrane-spanning DNA duplex on the lipids’ arrangement in the pore and presented how this arrangement can be remodelled depending on the hydrophilicity of the DNA design. Secondly, I have looked at the same system from the opposite perspective - studied and prevented the distortion of the transmembrane DNA construct induced by the surrounding lipids. Thirdly, I have evaluated the importance of ions in mediating DNA-lipid interactions, reporting analysis of two electrostatic phenomena: screening and bridging. Finally, utilizing a nanoengineered four-helix structure, I discussed surfactant’s influence on DNA membrane insertion efficiency, showing that aggregation of the nanostructures is one of the major factors determining their spontaneous membrane-spanning. While the understanding of phenomena in minimalistic systems is crucial for further development of complex pore-forming constructs, here I showed that even simple DNA nanostructures, when rationally designed, can mimic functionality of natural membrane proteins.EPSRC; Winton Programme for the Physics of Sustainabilit

Synaptic integration of transplanted fetal neurons into different neocortical environments

Brain repair strategies are becoming more promising as the approach of neuron transplantation has been tested in clinical settings, e.g., as therapy for Parkinson disease (PD). One important feature that transplanted neurons need to fulfill is their precise synaptic integration into the existing host brain network to truly reconstruct neuronal circuits. Brain-wide connectivity as well as functionality of grafted neurons was shown to be highly adequate. Transplanted neurons were proven to become functional and integrate with high specificity into the host cortical circuitry in a condition of upper layer neuron ablation. However, there is still little knowledge about brain-wide input connectivity of grafted neurons particularly concerning conditions of severe brain injury that goes along with reactive gliosis (brain trauma) or neurodegenerative diseases and aging with slow progression of synapse loss. Therefore, in the course of this PhD project I examined host-graft connectivity using monosynaptic rabies virus (RABV) tracing in cortical stab wound (SW) injury, intact, and inflamed cortical conditions in adult mice to evaluate if and to which extent these conditions integrate transplanted fetal neurons. In addition, I investigated graft integration in brain environments of progressive amyloidosis going along with synapse loss as observed in Alzheimer’s disease (AD) and of healthy aging to explore any influence of the aging brain environment per se. Indeed, in all these different host environments the grafted fetal neurons survived, differentiated, and integrated by forming connections with the correct host input regions. Surprisingly, brain-wide connectivity analysis showed that the grafts received excessive inputs from local neurons in the SW-injured, amyloid-plaque loaded, and aged environment. On the other hand, there was quantitatively fewer neuron integration in intact young control brains and in brains exposed to Lipopolysaccharide (LPS) induced inflammation as opposed to the massive input connections observed in the other conditions. Thus, new neurons integrate independent of prior neuron loss or mild reactive gliosis as grafted cells formed connections even in conditions where neuron loss did not occur. State-of-the-art proteome analysis using mass spectrometry (MS) revealed the protein compositions of these host cortical environments promoting excessive synaptic integration. This data provides important and highly relevant insights for the design of cell-based therapies for brain trauma and neurodegenerative diseases that go along with synapse loss. Understanding the mechanism that promote synaptic integration will open new avenues to modulate certain parameters in order to achieve adequate functional repair of lost neurons and synaptic connections

Post-transcriptional inhibition of human immunodeficiency virus type 1 (HIV-1) using combinatorial RNA interference (RNAi) expression vectors

Recent estimates indicate that globally there are over 33 million people infected with Human Immunodeficiency Virus type 1 (HIV-1). The epidemic is particularly severe in sub-Saharan Africa which accounts for 67% of all infected individuals and 72% of AIDS deaths in 2007. While current therapies, particularly in combination as a cocktail of highly active antiretroviral therapy (HAART), have had an important positive impact on the morbidity and mortality of HIV-related illness, there remain significant limitations. These include toxicities, resistance and the inability to eradicate a latent infection. In addition, most therapeutic agents have been developed to target HIV-1 subtype B, which affects individuals predominantly in Western Europe and North America. These concerns, along with ensuring patient compliance with treatment and the high cost of improved treatment regimens, have prompted the search for innovative and globally-effective therapies to eradicate HIV infection. More recently, gene therapy strategies based on the naturally-occurring RNA interference (RNAi) pathway has provided an exciting new mechanism to inhibit rogue gene expression. RNAi represents a set of highly conserved cellular pathways whereby double-stranded RNA (dsRNA) precursors are processed into shorter dsRNAs by the successive action of ribonucleases Drosha and Dicer. Processed, 21-23 nucleotide, short interfering RNAs (siRNAs) or antisense RNAs (asRNAs), associate with members of the Argonaute family of proteins to regulate gene expression at the transcriptional and post-transcriptional level. By exploiting the biogenesis of the endogenous mammalian RNAi pathway, several exogenous RNAi pathway mimics have been developed to inhibit unique sequences, including viral targets such as HIV. In the context of HIV, a combinatorial system that allows for the simultaneous suppression of multiple targets is important in preventing viral mutational escape of this rapidly evolving pathogen. The studies presented in this thesis add significantly to the newly emerging body of research on combinatorial RNAi strategies by focusing on the two novel technological approaches using mammalian expression systems. Both RNA Pol III-generated long-hairpin RNAs (lhRNAs) and RNA Pol II-generated polycistronic primary microRNAs (pri-miRNAs) were developed as systems for generating combinatorial RNAi precursors from single transcriptional units that induce post-transcriptional silencing of several highly conserved sequences of HIV-1. These included established therapeutic sites targeted to coding and non-coding regions of the HIV-1 long terminal repeat (LTR), Polymerase, Tat and Integrase. Expressed lhRNAs with ~63 bp duplex dsRNA regions and defined 5' and 3' termini were targeted to the transcribed region of the HIV 5' LTR and effectively suppressed two distinct sites within this region across both subtypes B and C HIV sequences. In addition, to assess whether lhRNAs could inhibit basal levels of HIV transcription, the lhRNAs were shown to suppress Tat-mediated (processive) and Tat-independent (non-processive) transcription when targeting episomal and integrated LTR-driven sequences. Portions of the lhRNAs that produced the most active siRNAs were dissected by using tiled LTR targets cloned into a luciferase reporter gene and by using northern blot analyses. Dicer-processing of expressed lhRNAs was shown to be most effective from the base of the duplex and decreased in efficiency towards the loop, suggesting that a gradient of siRNAs production is possible from a single lhRNA but with decreasing efficacy. This work laid the foundation for improved expressed lhRNAs whereby multiple unique anti-HIV siRNAs were produced from a single lhRNA. The second combinatorial RNAi strategy made use of RNA Pol II-expressed pri-miRNA mimics, where each mimic was derived from a different endogenous scaffold. Polycistronic transcripts consisting of four different pri-miRNA scaffolds and targeting four separate sites in HIV were tested in several combinatorial systems. The pri-miRNA backbone chosen was shown to dramatically affect the concentration and inhibitory efficacy of each generated effector strand, and this was largely independent of the sequence used. A strategy to combine four of the most effective pri-miRNA scaffolds into one expression cassette was developed and significant inhibition of an HIV infectious molecular clone as well as a wild type HIV isolate was demonstrated. Finally, in an attempt to uncover additional asRNAs capable of inducing inhibition via transcriptional gene silencing of the HIV LTR promoter, indiscriminate cell-wide gene activation was shown to occur as result of an unintended off-target effect. These observations demonstrated that caution should be exercised when interpreting RNA-induced gene activation results. Overall, this thesis provides a detailed description of the efficacy of two novel combinatorial RNAi approaches based on single promoter expression systems that are aimed at generating multiple RNAi effector sequences targeted to HIV. These approaches pave the way towards a better understanding of the efficacy of combinatorial RNAi and an effective and sustained gene therapy of HIV

Super-resolution mapping of receptor engagement during HIV entry

The plasma membrane (PM) serves as a major interface between the cell and extracellular stimuli. Studies indicate that the spatial organisation and dynamics of receptors correlate with the regulation of cellular responses. However, the nanoscale spatial organisation of specific receptor molecules on the surface of cells is not well understood primarily because these spatial events are beyond the resolving power of available tools. With the development in super-resolution microscopy and quantitative analysis approaches, it optimally poises me to address some of these questions. The human immunodeficiency virus type-1 (HIV-1) entry process is an ideal model for studying the functional correlation of the spatial organisation of receptors. The molecular interactions between HIV envelope glycoprotein (Env) and key receptors, CD4 and co-receptor CCR5/CXCR4, on the PM of target cells have been well characterised. However, the spatial organisation that receptors undergo upon HIV-1 binding remains unclear. In this project, I established a Single Molecule Localisation Microscopy (SMLM) based visualisation and quantitative analysis pipeline to characterise CD4 membrane organisation in CD4+ T cells, the main host cell target for HIV-1 infection. I found that prior to HIV engagement, CD4 and CCR5 molecules are organised in small distinct clusters across the PM. Upon HIV-1 engagement, I observed dynamic congregation and subsequent dispersal of virus-associated CD4 clusters within 10min. I further incorporated statistical modelling to show that this reorganisation is not random. This thesis provides one of the first nanoscale imaging and quantitative pipelines for visualising and quantifying membrane receptors. I showed that this quantitative approach provides a robust methodology for understanding the recruitment of HIV-1 receptors before the formation of a fusion pore. This methodology can be applied to the analyses of the nanoscale organisation of PM receptors to link the spatial organisation to function