Optimizing the Design of Oligonucleotides for Homology Directed Gene Targeting

BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA

Structural and Biochemical Studies of the Human DEAD-box Helicase Dbp5 and Nucleoporin Nup214 Involved in mRNA Export

The hallmark of eukaryotic evolution was the development of the nucleus in cells. This compartmentalization requires the nucleocytoplasmic transport of thousands of molecules. The gate into and out of the nucleus is the nuclear pore complex (NPC). One of the molecules that needs to be exported from the nucleus is messenger RNA (mRNA). mRNA associates with proteins in the nucleus forming a messenger ribonucleoprotein particle (mRNP). mRNPs bind to dedicated transport factors that facilitate movement through the NPC. One protein that associates to mRNPs is the helicase Dbp5, which belongs to the DEAD-box family of RNA helicases. Dbp5 is essential for mRNA export in both yeast and humans. It binds RNA and is concentrated and locally activated at the cytoplasmic side of the nuclear pore complex, where it interacts with the cytoplasmic nucleoporin Nup214. In my PhD work, I have determined the crystal structures of human Dbp5 bound to RNA and AMPPNP, and bound to Nup214. I designed and performed in vitro assays, which show that binding of Dbp5 to nucleic acid and to Nup214 is mutually exclusive. The interactions are mediated by conserved residues, implying a conserved recognition mechanism. These results suggest a framework for the consecutive steps leading to the release of mRNA at the final stages of nuclear export. More generally, they provide a paradigm for how binding of regulators can specifically inhibit DEAD-box proteins

The Mechanism of RecA Mediated DNA Patterning Interrogated by AFM

Over recent years, advancements in bottom - up construction technologies are enabling the creation of heterogeneous and functional materials. These approaches offer the potential to surpass the physical limitations in traditional top - down micromachining. Of these, bionanotechnological approaches that harness the inherent molecular recognition and self-assembling properties of biological molecules - such as Deoxyribose nucleic acid (DNA) - are arguably the most promising. Over recent years, the field of DNA nanotechnology has advanced rapidly, enabling the creation of arbitrary structures in two and three dimensions. These substrates act as adapters enabling the arrangement of functional components at the nano-scale to be interfaced with the macro-scale world. One approach to spatially address DNA nano-architectures is to harness the sequence specific homologous recombination mechanism of the E.coli protein Recombinase A (RecA). This protein mediates the alignment of a supplied single stranded DNA (ssDNA) with a subject double stranded DNA (dsDNA) where homology is shared, making this method inherently programmable. Despite several successful demonstrations of the artificial application of RecA, the underlying mechanism which orchestrates this interaction remains widely debated. The lack of clear understanding surrounding this critical biological mechanism stems from the in-direct approaches taken to interrogate it, to date. In response to this, the work presented in this thesis, attempts to answer the open biological questions surrounding RecA. Here, recent advances in high speed atomic force microscopy (HSAFM) and high resolution Atomic force microscopy (AFM) - using rapid-force-curve imaging - are applied to directly interrogate the homology searching mechanism of RecA. When taken together, these structural and functional insights will inform the future development of RecA mediated patterning approaches within complex DNA topologies

RecA-templated DNA scaffolds for selective site-specific assembly of nanoparticles for electronic devices

With today’s challenges in the electronic industry, novel alternative ap- proaches for manufacturing devices at nanoscale are being investigated. Using self-assembly, arguably has the best potential for nanostructures. DNA and proteins - some of the most important biomolecules use self- assembly extensively for natural functions. Chemical and structural pre- dictability of DNA and specificity of proteins promise a big potential for novel materials and could allow creation of structures controlled at nanoscale level. RecombinaseA - a DNA-binding protein has been used for controllable and predictable patterning of selected DNA sequences, opening the way to nanometre-scale DNA marking. However, protein patterning alone does not add any electric or other desired functionality to the DNA, there- fore additional modifications are neccessary. Furthermore, since biologi- cal molecules have transient functionality, system stability investigation is crucial for needed modification and subsequent usage. This project focused on RecA-patterned DNA modification for electric prop- erty addition. Thiolation and subsequent attachment of gold or magnetic nanoparticles to RecA protein present on DNA were investigated as a method for creating electrically conductive nanoscale objects. More specifically, at- tachment of gold nanoparticles throughout the whole patterned region of DNA and attachment of single nanoparticles at precise positions were looked into. The work successfully demonstrated that both nanoparticle deposition along the full length of RecA-coated DNA and specific single nanoparticle positioning is feasible. For investigating RecA-DNA stability, a system based on FRET was de- vised and used to analyse interaction kinetics. It was found that RecA-DNA complexes are fully formed in minutes and stay bound for hours. Specific configurations of the set-up showed distinct lack of signal, suggesting com- plicated interactions between the protein and patterned DNA. The project demonstrated through binding of NPs at specific locations and on the whole filament length that the system has potential for electronic applications and its stability is sufficient for processing times

TOWARDS ELUCIDATION OF THE MECHANISM OF BIOLOGICAL NANOMOTORS

Biological functions such as cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging, and cell entry all involve biomotor-driven DNA translocation. In the past, the ubiquitous biological nanomotors were classified into two categories: linear and rotation motors. In 2013, we discovered a third type of biomotor, revolving motor without rotation. The revolving motion is further found to be widespread among many biological systems. In addition, the detailed sequential action mechanism of the ATPase ring in the phi29 dsDNA packaging motor has been elucidated: ATP binding induces a conformational entropy alternation of ATPase to a high affinity toward dsDNA; ATP hydrolysis triggers another conformational entropy change in ATPase to a low DNA affinity, by which the dsDNA substrate is pushed toward an adjacent ATPase subunit. The subunit communication is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit, resulting in an asymmetrical hexameric organization. Continuation of this process promotes the movement and revolving of the dsDNA within the hexameric ATPase ring. Coordination of all the motor components facilitate the motion direction control of the viral DNA packaging motors, and make it unusually powerful and effective

Secondary somatic mutations restoring RAD51C and RAD51D associated with acquired resistance to the PARP inhibitor rucaparib in high-grade ovarian carcinoma

High-grade epithelial ovarian carcinomas (OC) containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and poly(ADP-ribose) polymerase inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pre-treatment and post-progression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed OC. In six of 12 pre-treatment biopsies, a truncation mutation in BRCA1, RAD51C or RAD51D was identified. In five of six paired post-progression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft (PDX), as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations

The physicist's guide to one of biotechnology's hottest new topics: CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.Comment: 75 pages, 15 figures, Physical Biology (2018

Single DNA conformations and biological function

From a nanoscience perspective, cellular processes and their reduced in vitro imitations provide extraordinary examples for highly robust few or single molecule reaction pathways. A prime example are biochemical reactions involving DNA molecules, and the coupling of these reactions to the physical conformations of DNA. In this review, we summarise recent results on the following phenomena: We investigate the biophysical properties of DNA-looping and the equilibrium configurations of DNA-knots, whose relevance to biological processes are increasingly appreciated. We discuss how random DNA-looping may be related to the efficiency of the target search process of proteins for their specific binding site on the DNA molecule. And we dwell on the spontaneous formation of intermittent DNA nanobubbles and their importance for biological processes, such as transcription initiation. The physical properties of DNA may indeed turn out to be particularly suitable for the use of DNA in nanosensing applications.Comment: 53 pages, 45 figures. Slightly revised version of a review article, that is going to appear in the J. Comput. Theoret. Nanoscience; some typos correcte

Self-Assembly from Milli- to Nanoscales: Methods and Applications

The design and fabrication techniques for microelectromechanical systems (MEMS) and nanodevices are progressing rapidly. However, due to material and process flow incompatibilities in the fabrication of sensors, actuators and electronic circuitry, a final packaging step is often necessary to integrate all components of a heterogeneous microsystem on a common substrate. Robotic pick-and-place, although accurate and reliable at larger scales, is a serial process that downscales unfavorably due to stiction problems, fragility and sheer number of components. Self-assembly, on the other hand, is parallel and can be used for device sizes ranging from millimeters to nanometers. In this review, the state-of-the-art in methods and applications for self-assembly is reviewed. Methods for assembling three-dimensional (3D) MEMS structures out of two-dimensional (2D) ones are described. The use of capillary forces for folding 2D plates into 3D structures, as well as assembling parts onto a common substrate or aggregating parts to each other into 2D or 3D structures, is discussed. Shape matching and guided assembly by magnetic forces and electric fields are also reviewed. Finally, colloidal self-assembly and DNA-based self-assembly, mainly used at the nanoscale, are surveyed, and aspects of theoretical modeling of stochastic assembly processes are discussed