End-joining long nucleic acid polymers

Many experiments involving nucleic acids require the hybridization and ligation of multiple DNA or RNA molecules to form a compound molecule. When one of the constituents is single stranded, however, the efficiency of ligation can be very low and requires significant individually tailored optimization. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically reduces dramatically. Here, we present a simple procedure to efficiently and specifically end-join two different nucleic acids using the well-known biotin–streptavidin linkage. We introduce a two-step approach, in which we initially bind only one molecule to streptavidin (STV). The second molecule is added only after complete removal of the unbound STV. This primarily forms heterodimers and nearly completely suppresses formation of unwanted homodimers. We demonstrate that the joining efficiency is 50 ± 25% and is insensitive to molecule length (up to at least 20 kb). Furthermore, our method eliminates the requirement for specific complementary overhangs and can therefore be applied to both DNA and RNA. Demonstrated examples of the method include the efficient end-joining of DNA to single-stranded and double-stranded RNA, and the joining of two double-stranded RNA molecules. End-joining of long nucleic acids using this procedure may find applications in bionanotechnology and in single-molecule experiments

Biomolecular Assembly of Gold Nanocrystals

Over the past ten years, methods have been developed to construct discrete nanostructures using nanocrystals and biomolecules. While these frequently consist of gold nanocrystals and DNA, semiconductor nanocrystals as well as antibodies and enzymes have also been used. One example of discrete nanostructures is dimers of gold nanocrystals linked together with complementary DNA. This type of nanostructure is also known as a nanocrystal molecule. Discrete nanostructures of this kind have a number of potential applications, from highly parallel self-assembly of electronics components and rapid read-out of DNA computations to biological imaging and a variety of bioassays. My research focused in three main areas. The first area, the refinement of electrophoresis as a purification and characterization method, included application of agarose gel electrophoresis to the purification of discrete gold nanocrystal/DNA conjugates and nanocrystal molecules, as well as development of a more detailed understanding of the hydrodynamic behavior of these materials in gels. The second area, the development of methods for quantitative analysis of transmission electron microscope data, used computer programs written to find pair correlations as well as higher order correlations. With these programs, it is possible to reliably locate and measure nanocrystal molecules in TEM images. The final area of research explored the use of DNA ligase in the formation of nanocrystal molecules. Synthesis of dimers of gold particles linked with a single strand of DNA possible through the use of DNA ligase opens the possibility for amplification of nanostructures in a manner similar to polymerase chain reaction. These three areas are discussed in the context of the work in the Alivisatos group, as well as the field as a whole

Monte Carlo simulation studies of DNA hybridization and DNA-directed nanoparticle assembly

A coarse-grained lattice model of DNA oligonucleotides is proposed to investigate how fundamental thermodynamic processes are encoded by the nucleobase sequence at the microscopic level, and to elucidate the general mechanisms by which single-stranded oligonucleotides hybridize to their complements either in solution or when tethered to nanoparticles. Molecular simulations based on a high-coordination cubic lattice are performed using the Monte Carlo method. The dependence of the model's thermal stability on sequence complementarity is shown to be qualitatively consistent with experiment and statistical mechanical models. From the analysis of the statistical distribution of base-paired states and of the associated free-energy landscapes, two general hybridization scenarios are found. For sequences that do not follow a two-state process, hybridization is weakly cooperative and proceeds in multiple sequential steps involving stable intermediates with increasing number of paired bases. In contrast, sequences that conform to two-state thermodynamics exhibit moderately rough landscapes, in which multiple metastable intermediates appear over broad free-energy barriers. These intermediates correspond to duplex species that bridge the configurational and energetic gaps between duplex and denatured states with minimal loss of conformational entropy, and lead to a strongly cooperative hybridization. Remarkably, two-state thermodynamic signatures are generally observed in both scenarios. The role of cooperativity in the assembly of nanoparticles tethered with model DNA oligonucleotides is similarly addressed with the Monte Carlo method, where nanoparticles are represented as finely discretized hard-core spheres on a cubic lattice. The energetic and structural mechanisms of self-assembling are investigated by simulating the aggregation of small "satellite" particles from the bulk onto a large "core" particle. A remarkable enhancement of the system's thermal stability is attained by increasing the number of strands per satellite particle available to hybridize with those on the core particle. This cooperative process is driven by the formation of multiple bridging duplexes under favorable conditions of reduced translational entropy and the resultant energetic compensation; this behavior rapidly weakens above a certain threshold of linker strands per satellite particle. Cooperativity also enhances the structural organization of the assemblies by systematically narrowing the radial distribution of the satellite particles bound the core

Transient Anions in Radiobiology and Radiotherapy: From Gaseous Biomolecules to Condensed Organic and Biomolecular Solids

This chapter focuses on the fundamental processes that govern interactions of low‐energy (1–30 eV) electrons with biological systems. These interactions have been investigated in the gas phase and within complex arrangements in the condensed phase. They often lead to the formation of transient molecular anions (TMAs), and their decay by autoionization or dissociation accompanied by bond dissociation. The damage caused to biomolecules via TMAs is emphasized in all sections. Such damage, which depends on a large number of factors, including electron energy, molecular environment, and type of biomolecule, and its physical and chemical interactions with radiosensitizing agents are extensively discussed. A majority of recent findings resulting from experimental and theoretical endeavors are presented. They encompass broad research areas to elucidate important roles of TMAs in irradiated biological systems, from the molecular level to nanoscale cellular dimensions. Fundamental aspects of TMA formation are stressed in this chapter, but many practical applications in a variety of radiation‐related fields such as radiobiology and radiotherapy are addressed

Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase

Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively poor helicase in its monomeric form, as has been reported. In summary, NS3h translocates on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein-displacement activity under identical conditions

Structural characterization and selective drug targeting of higher-order DNA G-quadruplex systems.

There is now substantial evidence that guanine-rich regions of DNA form non-B DNA structures known as G-quadruplexes in cells. G-quadruplexes (G4s) are tetraplex DNA structures that form amid four runs of guanines which are stabilized via Hoogsteen hydrogen bonding to form stacked tetrads. DNA G4s have roles in key genomic functions such as regulating gene expression, replication, and telomere homeostasis. Because of their apparent role in disease, G4s are now viewed as important molecular targets for anticancer therapeutics. To date, the structures of many important G4 systems have been solved by NMR or X-ray crystallographic techniques. Small molecules developed to target these structures have shown promising results in treating cancer in vitro and in vivo, however, these compounds commonly lack the selectivity required for clinical success. There is now evidence that long single-stranded G-rich regions can stack or otherwise interact intramolecularly to form G4-multimers, opening a new avenue for rational drug design. For a variety of reasons, G4 multimers are not amenable to NMR or X-ray crystallography. In the current dissertation, I apply a variety of biophysical techniques in an integrative structural biology (ISB) approach to determine the primary conformation of two disputed higher-order G4 systems: (1) the extended human telomere G-quadruplex and (2) the G4-multimer formed within the human telomerase reverse transcriptase (hTERT) gene core promoter. Using the higher-order human telomere structure in virtual drug discovery approaches I demonstrate that novel small molecule scaffolds can be identified which bind to this sequence in vitro. I subsequently summarize the current state of G-quadruplex focused virtual drug discovery in a review that highlights successes and pitfalls of in silico drug screens. I then present the results of a massive virtual drug discovery campaign targeting the hTERT core promoter G4 multimer and show that discovering selective small molecules that target its loops and grooves is feasible. Lastly, I demonstrate that one of these small molecules is effective in down-regulating hTERT transcription in breast cancer cells. Taken together, I present here a rigorous ISB platform that allows for the characterization of higher-order DNA G-quadruplex structures as unique targets for anticancer therapeutic discovery

Computational modeling of protein-ion binding and nucleic acids

Metal ions and nucleic acids are essential for a variety of biological functions. Metal ions play roles in enzyme catalysis, signal transduction and muscle contraction, and the stabilization of protein and nucleic acids structures. Nucleic acids are integral to gene expression and regulation. There are many unsolved questions regarding the function and thermodynamics of metal ions and nucleic acids. Molecular modeling has been an indispensable tool for microscopic understanding of biological processes. While tremendous success has been achieved for the modeling of proteins and organic molecules, it remains challenging to accurate model charged molecules, such as metal ions and nucleic acids. The difficulty mainly arises from the inadequate description of electrostatic interaction and polarization. In this work, accurate models for metal ions and nucleic acids based on AMOEBA polarizable force field were developed. These models along with advanced quantum mechanical methods were then used to study some practical problems. First, the principles underlying Ca²⁺/Mg²⁺ selectivity in ion-binding proteins were studied. It was shown that the Ca²⁺/Mg²⁺ selectivity can be explained by many-body polarization, which depends on the chemistry and geometry of the binding pocket. Second, an existing controversial question regarding the conduction mechanism of potassium channels was resolved by molecular dynamics (MD) simulations with AMOEBA. Contrary to previous beliefs, the conduction operates through nearly ion-saturated states. This mechanism is compatible with almost all existing experimental data. Third, free energy calculation with AMOEBA was used to predict the effect of chemical modifications on the stability of DNA-RNA hybrids, which has implications for the development of gene therapy. Overall, the AMOEBA polarizable force field significantly improves the accuracy for modeling of metal ions and nucleic acids. It is expected that application of polarizable force field will lead to more exciting findings on biological systems.Biomedical Engineerin

EXPLORING THE ROLE OF LIQUID CRYSTAL ORDERING OF DNA OLIGOMERS IN THE PREBIOTIC SYNTHESIS OF NUCLEIC ACIDS

The work in this thesis has been devoted to study the self-assembly of DNA oligomers in light of their possible relevance in the context of the formation of longer chains of nucleic acids in the prebiotic world. This conjecture is based on previous evidence of hierarchical self-assembly of short oligonucleotides in solution that provides mechanisms of self-selection. In such self-assembled structures, the 3\u2019 and 5\u2019 terminals are held in close contact, a condition that could act as a spontaneous template for the elongation of the chains in conditions favoring their chemical ligation. The work here described was aimed at testing this notion by various investigations targeting what appeared to be the most critical issues in this context. Accordingly, I acted in three main directions. First I investigated if LC ordering, and hence ligation templating, emerges even in solutions of oligomers having sequences chosen at random, and I determined a new extended phase diagram which includes the degree of randomness. Second, I determined what is the minimum oligomer length which enables the formation of LC phases and I found that LC phases can be found in solution of oligomers as short as 4-bases, and even with randomly chosed sequences. This last result was actually quite suprising and indicates the existence of a new regime for the self-assembly of ultra-short DNA chains. Third, I explored the influence of LC ordering of short DNA oligomers on non-enzymatic ligation reaction favored by the presence of a water-soluble condensing agent. I found a good yield for the polymerization of DNA oligomers both in isotropic than in LC phases, and polymerized chains up to 12 times longer than the initial length. I believe the work described in this thesis strengthens the notion that self-assembly of nucleic acids could indeed have been the key factor promoting the formation of long chains. These results shad a new light on the most obscure among the processes that enabled the emergence of life on the early Earth

Bio-Nano Robo-Mofos : Design and Synthesis of DNA Origami Nanostructures and Assembly of Nanobot Superstructures

In the field of bio-nanotechnology, molecules like DNA are repurposed as building materials for the construction of self-assembling nanostructures. The DNA origami method involves rationally coding many short synthetic DNA strands which ‘fold’ longer scaffold strands into precise, addressable structures for applications in areas like medicine, structural biology and molecular biophysics. DNA origami subunits are also used to explore fundamental principles of self-assembly, revealing insights into biology and expanding our control of matter at the nanoscale. But despite the usefulness of the method, DNA origami designs are limited in size by the length of scaffold strands, and in scope by the available tools needed to navigate the complex geometries of DNA nanostructures. My thesis addresses this in two ways: First, I present a set of principles for the design of DNA origami nanotubes, a class of strained structure with many applications. I parametrised variables related to nanotube design and created a computational tool to convert desired geometries into DNA strand layouts. I validated this via synthesis of various designs, including novel nanotubes with pleated walls, reconfigurable twist and varying diameter, characterising them with TEM, SAXS and MD simulations. This revealed insights into how design variables affect properties such as diameter and rigidity, and how global strain affects DNA nanostructures. Next, I present two schemes for assembling DNA origami subunits into self-limiting, open superstructures, exploring fundamental principles to control self-assembly while also overcoming DNA origami’s size limitations. The first is a strain accumulation scheme, which was explored theoretically and then embodied in a modular subunit with allosteric binding domains. With simulation and synthesis, I demonstrated that the subunit could structurally encode the extent of its own polymerisation. The second scheme is Vernier assembly, in which I showed that the combined geometries of two DNA origami subunits could determine the size of a superstructure and explored parameters important to maximise yield. Both studies provide guidance for future studies and applications which may require finite superstructures made from small numbers of unique components. Combined, the works in this thesis expand the design space for DNA-nanotechnology and fields beyond, enabling a range of biologically-inspired nanoscale autonomous modular formations, or ‘Bio-Nano Robo-Mofos’

The Interaction of Metal Complexes with G-quadruplex DNA

Un approccio computazionale è stato proposto per lo studio dell’interazione di complessi metallici di basi di Schiff con DNA. Nel capitolo 2, è stato investigato il meccanismo di azione di complessi di Nichel(II), Rame(II) e Zinco(II) con B e G-quadruplex DNA. Il G-quadruplex è una conformazione non canonica adottata da particolari sequenze ricche in guanina. Recentemente, è stata dimostrata la sua esistenza in cellule umane, in regioni telomeriche e non telomeriche, ed è stato proposto come un possibile target per una nuova categoria di agenti antineoplastici. I capitoli successivi sono basati su dati raccolti durante due periodi di ricerca all’estero. Nel capitolo 3, basato sugli studi eseguiti presso l’Università tecnica di Braunschweig, verrà mostrato come i campi di forza, oggigiorno di uso comune in Chimica/Biofisica Computazionale, siano in grado di riprodurre correttamente la stabilità relativa di G-quadruplex modello. Inoltre, è stata studiata in dettaglio l’interazione di una classe di leganti organici, noti G-quadruplex binders, con un modello di quadruplex parallelo. L’approccio computazionale ha messo in evidenza l’importanza del considerare esplicitamente la protonazione dei leganti. Nel capitolo 4, risultato di una COST Short-Term Scientific Mission in Francia presso l’Universitè de Lorraine, è descritta la procedura usata per riprodurre gli spettri di Dicroismo Circolare delle principali conformazioni dei G-quadruplex.A computational approach was proposed to study the binding and the stability of metal complex-nucleic acid supramolecular systems. In particular, the interaction of transition metal complexes with DNA structures named “G-quadruplexes” was considered. G-quadruplex conformations are present in telomeres and several oncogenes and they are involved in the inhibition of telomerase, a protein responsible for immortalization of cancer cells. The main purpose of the project was then to provide a computational tool to design chemical compounds able to selectively stabilize G-quadruplex structures