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Plasmids are circular double-stranded DNA and have been the workhorse of molecular biology. They can also be adopted by more complex organisms to influence how their cells behave. Importantly, this means they can be leveraged to study intricate molecular networks, and gene-function relationships, and hold promise as therapeutic devices. Thus, the ability to deliver and control their activity will not only advance their utility in research fields such as developmental and molecular biology but also expedite their translation to clinical settings. In this regard, light is a convenient stimulus as it is non-invasive, and its properties and application can be adjusted to minimise cellular damage. However, examples of light-controlled plasmid are limited, with the majority displaying poor light-dependent activation, or necessitating specific sequences that limit their use. This thesis outlines the development of a general strategy to deliver light-activated plasmids. This approach relied on photoblocking groups placed at the promoter, which could be removed in response to UV light. This strategy was used to control two ubiquitous bacterial and mammalian promoters. Cell-free reactions with the T7 promoter could be tightly repressed until the UV stimulus was applied. Likewise, light-regulated transcription and translation from the CMV promoter were also demonstrated in mammalian cells, although the photomodulation efficiency was more modest. However, this strategy failed to work on the tac and Sp6 promoters, which is likely due to the difference in binding interactions between the promoter and their cognate RNA polymerase partner. Thus, although this strategy worked well for the T7 promoter in cell-free reactions, the promoter dependency would mean that optimisation is required when the strategy is adopted to photocage alternative promoters. Furthermore, an outstanding challenge needs to be overcome before plasmids can be used for therapy: the inefficient delivery of large negatively charged molecules into cells. Initial studies in this regard were made, and suggestions to enable plasmid delivery were outlined. Unlike previous examples, this technology has the potential to be sequence agnostic, meaning it could be adaptable to any plasmid, greatly expanding its scope.

The G-quadruplex (G4) is a secondary DNA structure formed by the self-assembly of guanine nucleotides. Mapping G4 formation in the human genome shows that they are enriched in functionally important regions such as promoters, enhancers, transcription factor and architectural protein binding sites. However, the roles of G4s in biology remains elusive. Whilst G4s can exist in a folded or unfolded state, methods are required to ensure that endogenous G4 detection does not perturb G4 folding dynamics. In this work, G4-specific fluorescent probes are reported which enable single-molecule real-time visualisation of G4 structures in living cells. Single-molecule imaging was performed at nanomolar probe concentrations providing measurements of relative populations of G4s in living cells without global perturbation of G4 dynamics. Chemical trapping of the unfolded G4 state reveals that G4s alternate between their folded and unfolded state over a time course of 20 minutes. G4 formation in live cells was also shown to be cell-cycle dependent and disrupted by inhibition of transcription and replication. The observations presented here provide evidence to support dynamic formation of G4s in living cells. G4 visualisation methods were then deployed with the aim of investigating the potential involvement of G4s in 3D chromatin organisation. For this, G4 imaging was extended into 3D by super-resolution microscopy in fixed cells, and combined with single-cell Hi-C method. Hi-C is a fixed nucleus chromatin conformation capture technique that can simulate 3D chromatin structure via high-throughput sequencing of DNA strands in close proximity. 3D imaging of G4s has revealed their localisation throughout the nucleus volume with several regions showing clusters. When G4 mapping data were overlapped with single-cell Hi-C structures and G4s were visualised in 3D chromatin, this showed that G4s co-localised with the A compartment of the nucleus. By overlapping Hi-C structures with 3D super-resolved G4 images of the same single cells and population derived G4 sequencing data, the aim of this work is to establish a foundation to understand G4 relationships with respect to higher order 3D chromatin structure.

Cancer is one of the major causes of mortality nowadays, owing to the lack of accurate diagnostic and therapeutic techniques. DNA-based nanomaterials show extraordinary performance in cancer diagnosis and have applications in therapy, owing to their predictable secondary structures, small sizes, excellent biocompatibility, and programmability. Rolling circle amplification (RCA) has been identified as a promising technique to make DNA nanomaterials, including DNA hydrogels, origami, nanoribbons, nano scaffolds and DNA nanoflowers (DNFs). These DNA nanomaterials have potential in a wide variety of applications, involving drug delivery, in vivo imaging, electronic circuit fabrication and therapeutics. However, there was a lack of chemistry to expand their functionality, which greatly limited their applications. To address this, we developed a strategy involving commercially available modified nucleoside triphosphates in RCA, resulting in densely modified products. The modified RCA products can then be functionalised by targeting moieties such as aptamer and peptide, which enhance their binding ability towards specific cancer cells. The size of the RCA generated particles can be tuned through reaction time, Mg2+ concentration and the addition of polymers such as PLL and PEG. The PEGylated products can be labelled using the abovementioned method, leading to condensed fluorescent particles with better cellular uptake. Additionally, a nanocarrier system based on cyclic DNA was designed and evaluated for mRNA detection in cells. The untemplated cyclisation of DNA through CuAAC chemistry was efficient, and the multivalent system showed an improved binding towards target cancer cells. However, the internalisation of the system by cells needs to be improved. Entrapment of the system in subcellular compartments was observed, which significantly increased the background signal.

Chemically modified oligonucleotides find myriad applications in therapeutics and synthetic biology because they can be utilised to modulate biophysical or therapeutic properties, install fluorophores for detection, or to allow orthogonal reactivity through additional functional groups. Whilst oligonucleotides can be principally modified anywhere at their nucleotide subunits, the presented work investigates modifications of the oligonucleotide backbone for therapeutic applications and synthetic biology. Antisense oligonucleotides (ASOs) have become an emerging therapeutic approach to modulate protein expression at the mRNA level to combat hard-to-treat diseases. Stability against nucleolytic degradation, good target hybridisation properties, and efficient delivery into the cell are essential. The chemical modification of the hydrolysable, anionic phosphodiester backbone holds great potential for protection against phosphodiesterases, and to reduce the polyanionic charges for enhanced cellular uptake. In the presented work, two charge-neutral backbone surrogates-the carbamate and amide internucleoside linkages-were investigated for their ability to confer drug-like properties by replacing the natural phosphodiester backbone. Combinations with additional bicyclic sugar modifications and different designs of the ASO segments were explored. The antisense assessment criteria comprised their general hybridisation properties with an RNA target, their stability against nucleolytic degradation, and their potential for enhanced cellular uptake. Backbone-modified oligonucleotides are also versatile constructs for synthetic biology as exemplified by the construction of synthetic genetic polymers, ribozymes, aptamers, or gene editing systems. Amongst those, artificial genetic polymers can be applied for gene synthesis, sequencing, or the production of nanomaterials. A crucial element for backbone modifications in genetic templates is their compatibility to be read by polymerases. In this context, triazole-based internucleoside linkages have been extensively studied as promising replication compatible phosphodiester mimics. This thesis presents a novel triazole backbone structure and its assessment for replication compatibility in terms of continuation, speed, and correctness of the read-through by DNA polymerases.

The selection of 5' splice sites in pre-mRNA transcripts containing multiple splice sites is poorly understood. RNA flexibility and how it affects splice site selection has not currently been investigated on a molecular level. However, secondary structures in proximity to the splice site and RNA-protein/small molecule binding events has been shown to shift splicing using ensemble fluorescence methods. Evidence suggests that using small molecules which target cancerous mutations caused by incorrectly spliced genes such as induced myeloid leukaemia cell differentiation and β-cell lymphoma proteins may have therapeutic application. Single molecule experiments are not limited by ensemble averaging and so can be used to study the conformational flexibility of individual RNA molecules in functional splicing conditions. Single molecule fluorescence resonance energy transfer was used in this thesis to monitor the conformational flexibility of different nucleic acids including DNA (Holliday junction and molecular beacon) and RNA (adenovirus and β-cell lymphoma sequences) structures. The experiments were performed under different conditions using various methods of surface passivation and surface tethering (direct tethering and encapsulation in lipid vesicles). The experiments outlined here indicate two main things: firstly, that monitoring nucleic acids which are freely diffusing in lipid vesicles (100 nm) is more representative of physiological conditions. Single molecules in lipid vesicles demonstrated more homogeneous conformational dynamics than those samples which had been tethered directly to the imaging surface. Secondly, we have shown that the G-quadruplex putative to the Xs splice site in the β-cell lymphoma gene forms and is stabilised by ellipticine derivative, GQC-05 in HeLa cell nuclear extract. This suggests that the stabilisation of the quadruplex and alteration to mRNA flexibility upon binding likely underlies its ability to shift splicing of β-cell lymphoma, from an anti- to a pro-apoptotic isoform. The single molecule results presented here complement previous literature and combined suggest that GQC-05 is a candidate pro-drug molecule for the therapeutic relief of tumours.

Modified backbones are versatile modifications which can improve biological half-lives, have good structural biocompatibility and can alter the biophysical properties of oligonucleotides. This work presents the synthesis and biophysical properties of oligonucleotides modified with carbamate-locked nucleic acids (CBM-LNA) for antisense applications, the crystallisation and structural analysis of amide-locked nucleic acids (LNA-amides) and explores peptide translation using modified triazole backbones in circular mRNAs (Tz-circRNA). From these studies it is shown that CBM-LNAs present viable templates for potential antisense applications, that LNA-amides are good structural mimics as phosphodiesters and that Tz-circRNAs are viable templates for protein expression. Carbamate backbones are four-atom, charge neutral linkages that were previously found to be duplex destabilising and unsuitable for antisense applications. In this work, we combine the enzymatic stability of the CBM backbone with the thermodynamic stability of locked nucleic acids (LNA). CBM-LNA phosphoramidite dimers were synthesised and incorporated into oligonucleotide sequences. Biophysical characterisation using UV melting, showed that LNA-CBM1-LNA modifications were able to restore duplex stability against RNA targets and that LNA-CBMs have enhanced enzymatic resistance. Compared to previous CBM modifications, LNA-CBMs now present suitable modifications for potential antisense applications. LNA-amides have good thermodynamic properties which makes them suitable candidates for therapeutic applications. The aim of our work is to show that LNA-amides are good structural mimics as phosphodiesters and suggest the features that provide LNA-amides with good biophysical properties. XRD crystallography was used to obtain three different LNA-amide modified oligonucleotides between 2.5-2.8 Å resolution. Analysis of these structures showed that LNA-amides are structurally alike to phosphodiesters and that they cause minimal duplex disruption. The amide and LNA modifications were found to work harmoniously together. To provide good duplex stability, modified backbones should have good flexibility towards their 5ˈ-end and should consist of four-atoms in length if they contain a carbonyl functionality. Triazole backbones have good biocompatibility for replication and transcription mechanisms. In this work we aim to complete the central dogma and translate peptides using triazole modified circular mRNA templates. This will allow for the chemical synthesis of circular mRNAs > 100 nt without the need for enzymes, plasmid preparation or bacterial cell culture. Linear precursor RNAs were modified with 5ˈ-azide and 3ˈ-alkyne functional groups. These were then subjected to the CuAAC reaction to form a triazole linkage contained within the coding region of a circular mRNA. Encoded with FLAG epitopes, the triazole circular RNAs were used in cell free prokaryotic protein translation and analysed by immunoblotting techniques. From these results it is shown that triazoles can be successfully translated by the prokaryotic ribosome and yield up to 50% as much peptide as an unmodified circular RNA.

Fluorogenic oligonucleotide-based systems are important tools for sequence-specific DNA targeting. Fluorescence systems use the emission of a photon from an excited molecule (fluorophore) to produce a detectable signal. It is a highly sensitive process and its versatility has expanded its use to DNA targeting probes. The aim of the work herein is to investigate the design and synthesis of fluorogenic oligonucleotide-based systems specifically for targeting double-stranded DNA. Triplex-forming oligonucleotides can bind in the major groove of dsDNA through Hoogsteen hydrogen bonding to form triplexes (triple helixes). While other methods of dsDNA detection rely on denaturation of the double-stranded target, triplexes do not, and, hence, they provide a versatile approach to DNA recognition. However, the instability of triplexes, especially at neutral pH, limits their applications. The incorporation of a fluorogenic intercalator, thiazole orange, covalently attached to the TFO provides a triplex-stabilising moiety. The stabilising effect of thiazole orange is additive; multiple incorporations can produce triplexes with increased stability of +50 °C even at neutral pH. The extreme stability provided from the modified fluorogenic TFOs allows increased duplex binding affinity and can compensate for the limited target range of TFOs. CRISPR-Cas is a programmable biological research tool that is invaluable in gene- editing and genomic imaging technologies. The introduction of fluorogenic moieties both on the dCas9 enzyme and in guide RNA is crucial for imaging but often leads to high background fluorescence in biological systems. By modifying the gRNA to contain an 'on/off' fluorogenic system, genomic imaging in live cells of repetitive elements is improved and is cross-validated by GFP-tagged dCas9. The efficient synthesis of long, modified RNA is fundamental, particularly in CRISPR research. The introduction of an azide moiety into RNA to provide a means of 'clicking' modified RNA strands together is an important potential approach. However, it is limited by the protecting groups used for RNA and, in turn, this hinders the length of efficient RNA synthesis. The synthesis of a clickable moiety compatible with 2'-O-TC RNA chemistry is investigated as it could provide a simple and cost- effective route to modified guide RNA and could be instrumental in applications such as modified guide RNA library generation. Overall, the aims of this study were to synthesise and develop fluorogenic oligonucleotide-based systems and investigate their potential applications.

Nucleic acid analogues are very attractive molecules for therapeutic applications as in principle they can specifically inhibit the expression of virtually any gene through antisense or RNA interference pathways. For this purpose, nucleic acid analogues with modified backbones have advantageous properties compared to their natural counterparts. In particular they can provide better resistance to nucleases as well as improving cellular uptake. However, radically changing the nucleic acid backbone while maintaining a good affinity for a mRNA or DNA target remains challenging. Developing synthetic strategies to conveniently access new modified backbones that could be used in antisense oligonucleotides is therefore essential. With this in mind a new solid-phase method to efficiently functionalise an amine-modified DNA backbone has been developed. Using this approach, three new oligonucleotide modified backbones have been synthesised, and their hybridisation properties have been investigated. Modified DNA backbones are also potentially useful in the development of new methods for chemical-ligation of oligonucleotides. This approach could allow the large-scale de novo synthesis of genes containing site-specific chemical modifications including epigenetic signals. Such constructs represent challenging targets in medicinal and biotechnology applications. An important class of modified DNA linkages containing a triazole ring developed in our research group has been further investigated through the synthesis of two new triazole modified backbones. The synthesis of a DNA amide backbone with favourable duplex stability was also performed. A detailed study of the biophysical properties of the above modified backbones including their performance as templates for various DNA polymerases has been carried out.

CRISPR-Cas, a bacterial adaptive immune system against viruses, has been repurposed into a biological research tool that is invaluable due to its simplicity and programmability. Composed of a Cas endonuclease that is directed to its nucleic acid target by a guide RNA (gRNA), CRISPR has been used for genome editing, live-cell imaging and even diagnostics. In this work, the goal is to chemically modify gRNAs to expand CRISPR functionality. A 'split-and-click' strategy for generation of Cas9 gRNAs via CuAAC 'click' ligation of shorter oligonucleotides is developed. The approach provides a simple and cost-effective route to individual or pools of single guide RNAs (sgRNAs), while providing scope for further chemical modification to improve sgRNA activity. The click sgRNAs are functional both in vitro and in cells, demonstrating artificial linkages can be tolerated in functionally critical regions of sgRNA. To illustrate the capabilities of the system, work towards an on-field forensic analysis of Short Tandem Repeats (STR) is described using CRISPR, Ramified Rolling Circle Amplification (RAM) and Oxford Nanopore sequencing. sgRNAs were also tagged with small molecule fluorophores to provide greater flexibility in colour choice and expand the colour range for live-cell imaging. Imaging of repetitive elements of telomeres is feasible with these constructs if GFP-tagged dCas9 is used for cross validation; due to high cytoplasmic background fluorescence, methods to turn on fluorescence in the nucleus are also explored. Finally, two CRISPR-AsCas12a gRNAs are chemically ligated to yield a 3'-3' dual gRNA with the aim of altering the DNA cleavage pattern of the Cas12a protein. Chemical ligation in this context impaired gRNA activity in vitro, however unexpected single-stranded DNase activity is described for AsCas12a.

Macromolecular X-ray crystallography (MX) is currently the dominant technique for the structural eluci- dation of macromolecules at near atomic resolution. However, the progression and deleterious effects of radiation damage remains a major limiting factor in the success of diffraction data collection and subsequent structural solution at modern third generation synchrotron facilities. For experiments conducted at 100 K, protein specific damage to particular amino acids has been widely reported at doses of just several MGy, before any observable decay in average diffraction intensities. When undetected, such artefacts of X-ray irradiation can lead to significant modelling errors in protein structures, and ultimately the failure to derive the correct biological function from a model. It is thus vital to develop tools to help MX experimenters to detect and correct for such damage events. This thesis presents the development of an automated program, RIDL, which is designed to objectively quantify radiation-induced changes to electron density at individual atoms, based on Fobs,n − Fobs,1 Fourier difference maps between different dose states for a single crystal. The high-throughput RIDL program developed in this work provides the ability to systematically investigate a wide range of macromolecular systems. To date, damage to the broad class of nucleic acids and nucleoprotein complexes has remained largely uncharacterised, and it is unclear how radiation damage will disrupt the validity of such models derived from MX experiments. This thesis presents the first systematic investigations on a range of nucleic acid, protein-RNA and protein-DNA complex case studies. In general, it is concluded that nucleic acids are highly robust to radiation damage effects at 100K, relative to control protein counterparts across the tested systems. For protein crystals at 100K, cleavage of the phenolic C-O bond in tyrosine has disseminated through the MX radiation damage literature as a dominant specific damage event at 100K, despite the absence of any energetically favourable cleavage mechanism. To clarify the radiation susceptibility of tyrosine, this thesis presents a systematic investigation on radiation damage to tyrosine in a wide range of MX protein radiation damage series retrieved from the Protein Data Bank. It is concluded that the tyrosine C-O bond remains intact following X-ray irradiation, however the aromatic side-group can undergo radiation-induced displacement. This thesis also presents further applications of the RIDL program. A protocol is introduced to calculate explicit half-dose values for the electron density at individual atoms to decay to half of their initial value at zero absorbed dose. In addition, a methodology is developed to detect radiation-induced changes to electron density occurring over the course of the collection of a single MX dataset of diffraction images, all of which are required for structural solution. These protocols aim to advise experimenters of when previously-undetected site-specific damage effects may have corrupted the quality of their macromolecular model. Overall, the work in this thesis is highly applicable to both the future understanding of radiation damage in macromolecular structures, as well as of interest to the wider crystallographic community.

This thesis describes the synthesis of derivatives of 2'-ethynyl-2'-deoxynucleosides, and their conversion into phosphoramidite building blocks for DNA synthesis. Herein is also outlined the preparation of azide-bearing nitroxyl radicals. After site-selective incorporation of 2'-alkynylnucleosides into DNA using a standard solid-supported phosphoramidite protocol, the modified oligonucleotides were spin labelled with the azide-bearing nitroxyl spin labels via the Cu(I)-catalysed azide-alkyne [3+2] Huisgen cycloaddition (CuAAC or 'click' reaction). The destabilising effect of the spin labelling was determined by UV denaturation studies and circular dichroism, and the spin labelled DNA was investigated by EPR spectroscopy. It was found that this novel site-directed spin labelling strategy afforded conformationally restricted systems, and that the structure of the spin label exerts a significant influence on the results.

NMR chemical shifts have an extremely high information content on the behaviour of macromolecules, owing to their non-trivial dependence on myriads of structural and environmental factors. Although such complex dependence creates an initial barrier for their use for the characterisation of the structures of protein and nucleic acids, recent developments in prediction methodologies and their successful implementation in resolving the structures of these molecules have clearly demonstrated that such barrier can be crossed. Furthermore, the significance of chemical shifts as useful observables in their own right has been substantially increased since the development of the NMR techniques to study low populated 'excited' states of biomolecules. This work is aimed at increasing our understanding of the multiple factors that affect chemical shifts in proteins and nucleic acids, and at developing high-quality chemical shift predictors for atom types that so far have largely escaped the attention in chemical shift restrained molecular dynamics simulations. A general approach is developed to optimise the models for structure-based chemical shift prediction, which is then used to construct CH3Shift and ArShift chemical shift predictors for the nuclei of protein side-chain methyl and aromatic moieties. These results have the potential of making a significant impact in structural biology, in particular when taking into account the advent of recent techniques for specific isotope labelling of protein side-chain atoms, which make large biomolecules accessible to NMR techniques. Through their incorporation as restraints in molecular dynamics simulations, the chemical shifts predicted by the approach described in this work create the opportunity of studying the structure and dynamics of proteins in a wide range of native and non-native states in order to characterise the mechanisms underlying the function and dysfunction of these molecules.

HIV-1 has evolved a number of strategies in response to current anti-retroviral drugs and the selection pressure of humoral and cellular immunity. In particular, R5 viral strains that are essential for AIDS pathogenesis are very resistant to neutralization by antibodies. Therefore, the aim of this thesis was to develop synthetic nucleic acid ligands, aptamers, against gp120 of an R5 strain of HIV-1, with a view of using aptamers as novel neutralization molecules and analytical tools to study HIV-1 entry into target cells. The central hypothesis of this thesis was that aptamers by virtue of their small size and slow dissociation rates, compared to antibodies, would easily access and bind occluded gp120 neutralization sites. Using the SELEX protocol and SPR technology, I isolated 2'-Fluoro-pyrimidine-RNA aptamers against HIV-lBa-L monomeric gp120. Most of these aptamers not only bound gp120 with high affinities but also neutralized R5 primary isolates in human PBMC by 1,000 to 100,000-fold, truly unprecedented when compared with natural ligands such as antibodies. Some aptamers, like B4, defined a conserved site of gp120 that could not mutate to escape neutralization following stringent selection, in vitro, for breakthrough virus. This was consistent with subsequent findings that B4 aptatope (binding site) overlaps a poorly immunogenic but highly conserved CD4-induced epitope as determined by competition with 17b and 48d mAbs that map to this neutralization epitope on the gp120. This study was thus the first of its kind to describe neutralization of HIV-1 primary isolates by a ligand against the CD4-induced epitope. Most intriguing, although B4 potently neutralized HIV-1Ba-L infection in PBMC, which is a mixed T cell and macrophage population, it modestly neutralized infection of the same virus in a purified culture of macrophages. These findings are intriguing in that they suggest that aptamers could be used to dissect unique sites on the virus that interact with target cell surface in ways that have not been revealed heretofore, and would help understand better HIV-1 entry pathways, especially in macrophages. Thus neutralizing aptamers such as these could be exploited to provide leads in developing alternative anti-HIV-1 drugs and a deeper understanding of the molecular interactions between the virus and its host cell.

Genomic DNA fragments encoding the silent type B neurotoxin gene from Clostridium botulinum NCTC 2916 have been cloned and the complete nucleotide sequence determined. The translated sequence revealed that the gene encoded a neurotoxin which was closely related to type B neurotoxin genes from Group I Clostridium botulinum. However among the nucleotide sequence differences, aG to T transition has interrupted the coding sequence with the formation of a stop codon. In addition the deletion of an adenine residue has resulted in a frame-shift mutation. Analysis of the DNA sequence contiguous with the silent type B neurotoxin gene revealed the presence of a gene encoding a Nontoxic-Nonhaemagglutinin protein which appears to share a bicistronic mRNA transcript with the type B neurotoxin gene. In the reverse orientation, the partial sequence of a gene encoding a haemagglutinin protein was found, typical of type A and B botulinal neurotoxin complexes. Separating the genes encoding the 'components of the neurotoxin complex was a gene of 178 amino acids which possessed features commonly associated with transcriptional factors. To facilitate the in vivo study of botulinal neurotoxin complex regulation, a gene transfer system using clostridial components has been developed. The minimal replicon of the cryptic plasmid pCB 102 from Clostridium butyricum NCIB 7423 was located to 1.6 kb DNA fragment by deletion analysis, enabling the identification of hitherto undiscovered putative ORFs and secondary structures, consistent with a replicative function. The replicon has been incorporated in to a number of Escherichia coli vectors resulting in a versatile series of shuttle vectors which have demonstrated high structural and segregational stabilities in a heterologous host Clostridium beyerinckii NCI NIB 8052. Gene transfer of a Group I Clostridium botulinum type A strain was demonstrated with a representative pCB 102-derived shuttle vector, pMTL540E. In addition, a 5.9 kb plasmid indigenous to C. hotulimun NCTC 2916 was cloned and the complete nucleotide sequence determined. Eight putative ORFs have been identified, including a putative replication protein and recombinase.

This work presents an examination of the subcellular localization of the major myelin proteins of oligodendrocytes, and the messages that encode these proteins. The association of these messages with the cytoskeleton is also examined. All messages investigated show some degree of association with the cytoskeleton; Indeed messages encoding actin and tubulin are found exclusively bound to the cytoskeleton. In general a specific myelin message and its protein counterpart are found in the same area of the cell. This suggests that these messages are translated close to their targetted site. In most Instances the messages being probed by in situ hybridization are being sought for the first time in cells grown in tissue culture, and in some instances messages are being probed for the first time ever. In addition, the messages encoding DM-20 and PLP are found in different areas of the cell. As these are the products of a single gene, this suggests that there is a significant role to be played by differential splicing in individual cells. The role of cytoske1eton-mRNA association in the targetting and assembly of myelin proteins is discussed.

The next generation of anticancer agents will emerge from rationally designed nanostructured materials. This work involved the synthesis and characterization of novel hollow DNA-conjugated gold nanoparticles (DNA-AuNPs) for controlled drug delivery. Polyethyleneimine (PEI) was bound to citrate-capped AuNPs, forming polymer-shell nanoparticles. Dissolution of the gold core via iodine formed hollow core polymeric nanoparticles (HCPPs) and a high density of DNA (85 molecules/particle) containing daunorubicin was conjugated. Particles were spherical with an average diameter of 105.7±17.3 nm and zeta potential of 20.4±3.54 mV. We hypothesize the DNA backbone electrostatically condensed to the primary amines on the surface of the particle toroidally, weaving itself beneath. HCPPs released 225±44.6 mg more drug/cm3 than PEI capped AuNPs and showed release over 12 hours, an order of magnitude longer than PEI capped AuNPs. No change in release regarding DNA strand length or composition was noted. Increasing ionic concentration increased drug intercalation 64%, and intercalating drug to the DNA prior to nanoparticle conjugation resulted in 32% more drug loaded. A 10-fold decrease in the amine/phosphate ratio led to a 61% increase in drug intercalation. HCPPs did not aggregate in albumin and increased DNA stability and functionality when exposed to nuclease more than DNA conjugated gold nanoparticles. Drug delivery via HCPPs have promise to be more clinically efficacious compared to current nanoparticle designs.

Base editors are tools that chemically modify the nucleobases of DNA and RNA in a programmable manner, allowing for genome, epigenome, and transcriptome editing in live cells. These tools can be used to introduce specific base transitions in DNA or RNA, manipulate methylation patterns in the epigenome, and create genetically encoded libraries in target genes. These various functions can be used to modulate every aspect of the central dogma. The efficiency and precision of base editors makes them useful in both basic research and in the development of new therapies. The adenosine base editor (ABE) was developed by evolving the RNA-modifying enzyme TadA to also accept DNA as substrate. This suggests that the base editing tool kit can be expanded to use many of the naturally occurring RNA-modifications as intermediates. However, current base editors remain limited to deaminases, suggesting that more complex chemical reactions face additional barriers. To better inform the creation of new base editors, we set out to better understand the molecular mechanisms of the wild-type TadA enzyme (used in ABE0.1) and the DNA-modifying variants developed for base editing (ABE7.10, ABE8e, ABE8.20, etc.). First, we observe that ABE0.1 shows high Cas9-independent, off-target RNA-editing at UACG motifs in accessible loops. In chapter 3, we ask if this discovery also extends to DNA and find that ABE0.1 can perform targeted DNA-editing at TACG motifs. This editing is inefficient though and may be easily missed, so we leveraged this discovery to develop a fluorescent reporter of base editing that greatly increases our ability to detect low-levels of base editing. We finish out the chapter by using this fluorescent reporter to better understand the sequence-dependent editing of ABE0.1 and evolved ABEs. Finally, we apply this new understanding of an early base editor to develop more sequence-optimized fluorescent reporters to: detect DNA editing by RNA-modifying enzymes that could be used for base editing in chapters 4 and 5, develop a protocol for evolving new base editors in mammalian cells in chapter 6, and developing an outreach experiment to introduce base editing to high school students in chapter 7. Through the experiments in each of these sections, we gain a better understanding of how the sequence recognition properties of RNA-modifying enzymes can be leveraged to develop new base editors.

Nucleic acid compaction is ubiquitous in biology, from chromatin and genome packing to RNA folding. Nature achieves these different processes in several different ways, but in all cases nucleic acid helices must be brought into contact or close proximity, often at a substantial free energy penalty. We developed quantitative disulfide crosslinking and tethered FRET techniques to measure the energetics of these fundamental interactions, and to explore the factors influencing them. We used a bimolecular crosslinking assay to measure the electrostatic repulsion governing unmediated encounters between DNA helices. By varying the length of the crosslinker probes, we established the size of the ion atmosphere surrounding the helices under different ionic conditions. We complemented these results with a FRET assay of the relative orientations of two DNA helices tethered by a short PEG linker. In the presence of Na⁺ or Mg²⁺, both bimolecular crosslinking and tethered FRET revealed a transition from a low salt regime dominated by electrostatic repulsion to a high salt electrically screened regime dominated by thermal motion. With Co(NH₃)₆³⁺, both assays revealed a transition to a net attractive regime with dilute phase characteristics consistent with previously observed Co-induced DNA condensates, suggesting that such condensates are the result of pairwise interhelical attraction. We also used an intramolecular crosslinking assay to measure the energetics of sharply bent conformations for a selection of RNA two-helix junctions. For model oligo-U bulges and junctions (Uₓ₌₂₋₇Uᵧ₌₀₋₃), junctions with more unpaired nucleotides on the longer strand (Uₓ) displayed more frequent sharp bending, with little dependence on the length of the shorter strand (Uᵧ). We probed different bent conformations by varying the crosslinker positions, identifying conformational preferences up to ~3 kcal/mol among different bent states. We also examined four naturally-occurring junction sequences (two from ribosomal RNAs and two from group I introns) that are sharply bent in their native folds. We found that these natural junction sequences do not selectively stabilize specific bent conformations corresponding to their native folds, instead exhibiting conformational lability, and behaving much like their oligo-U topological counterparts.

A central goal of biomolecular engineering is the construction of tools to manipulate nanoscale processes. DNA has proved to be a programmable material suited for this task. DNA strand displacement reactions can be designed to process chemical information in the form of concentrations and sequences. DNA nanotechnology has thus far produced devices for the detection of disease biomarkers, performed computation on chemical inputs, powered mechanical action at both the nanoscale and the macroscale, and assembled precise sub-micron structures from the bottom up. This dissertation addresses three main topics. First, we develop predictive models for non-canonical nucleic acid hybridization that enable rational design. Second, we show how rationally designed DNA strand displacement reactions can be used to perform computations on information stored in DNA. Third, we present nucleic acid computation with both strand displacement and transcription and discuss strategies for facilitating the scale up of networks. Finally, we discuss data storage in nucleic acid variants in the appendix. Rational design of DNA circuits and structures is possible because the thermodynamics of DNA and RNA hybridization can be approximated using a nearest-neighbor model. The parameters of this model are typically experimentally determined through the hyperchromism of denatured nucleic acids. This is measured through low-throughput UV-Vis spectrophotometry melting experiments that require a sizable amount of duplexes for a large set of sequences. For non-canonical nucleic acids or non-standard interactions, this characterization can be prohibitively costly and time consuming. Initially, we considered repurposing a next-generation sequencing (NGS) platform for high-throughput mapping of nucleic acid hybridization across a large sequence space; however, we found that the platform is suitable for mapping protein-nucleic acid interactions but not nucleic acid-nucleic acid hybridization due to its dynamic range. We then assessed whether high-resolution melting (HRM) can be used as a rapid method for determining approximate model parameters and found that HRM models can predict relative stabilities between duplexes of different sequences. Using this method, we developed a predictive model for phosphorothioate DNA which we then applied to the design of a phosphorothioate-modified catalytic hairpin assembly circuit. DNA strand displacement reactions can be used not only to manipulate chemical information in the form of concentration, but also to read and write to more permanent forms of information, such as sequence and secondary structure. We developed and demonstrated a DNA data storage scheme that enables in-memory computation. DNA is a promising data storage medium for meeting today's rapidly growing data storage needs; however, because computation on the stored data is usually performed in silico, strands must be sequenced and re-synthesized at every read-write cycle. Our scheme circumvents the bottleneck of de novo oligonucleotide synthesis by updating information using strand displacement cascades that result in sequence changes readable by NGS. We experimentally demonstrated two algorithms - binary counting and cellular automaton Rule 110 - and additionally showed that biologically-occurring DNA sequences without sequence design can be repurposed for storage and computation. Our scheme is capable of computation on multiple data in parallel, as well as random access and sequential computation, allowing for scaled up storage. Programmable chemical computation is also possible with enzymatic reactions such as transcription. Catalytic activity from enzymes has the potential to simplify circuit design and produce biologically potent signals. Practical concerns to expanding chemical computation circuits such as transcription networks include limited readout of signals and time-consuming purification. We addressed these concerns by expanding on previous efforts to build scalable in vitro transcription networks. We updated a single-stranded inhibitory transcription switch design for compatibility with multiplexed NGS readout and developed an analogous single-stranded switch that is activated by nucleic acid signals.

This dissertation details the use of computational methods to understand the effect that cancer-related mutations have on proteins that complex with nucleic acids. Firstly, we perform molecular dynamics (MD) simulations of various mutations in DNA polymerase κ (pol κ). Through an experimental collaboration, we classify the mutations as more or less active than the wild type complex, depending upon the incoming nucleotide triphosphate. From these classifications we use quantum mechanics/molecular mechanics (QM/MM) to explore the reaction mechanism. Preliminary analysis points to a novel method for nucleotide addition in pol κ. Secondly, we study the ten-eleven translocation 2 (TET2) enzyme in various contexts. We find that the identities of both the substrate and complementary strands (or lack thereof) are crucial for maintaining the complex structure. Separately, we find that point mutations within the protein can affect structural features throughout the complex, only at distal sites, or only within the active site. The mutation's position within the complex alone is not indicative of its impact. Thirdly, we share a new method that combines direct coupling analysis and MD to predict potential rescue mutations using poly(ADP-ribose) polymerase 1 as a model enzyme. Fourthly, we perform MD simulations of mutations in the protection of telomeres 1 (POT1) enzyme. The investigated variants modify the POT1-ssDNA complex dynamics and protein—DNA interactions. Fifthly, we investigate the incorporation of remdesivir and other nucleotide analogue prodrugs into the protein-RNA complex of severe acute respiratory syndrome-coronavirus 2 RNA-dependent RNA polymerase. We find evidence for destabilization throughout the complex and differences in inter-subunit communication for most of the incorporation patterns studied. Finally, we share a method for determining a minimum active region for QM/MM simulations. The method is validated using 4-oxalocrotonate, TET2, and DNA polymerase λ as test cases.

G-quadruplexes (GQs) are highly stable noncanonical nucleic acid structures that form in the DNA of human cells and play fundamental roles in maintaining genomic stability and regulating gene expression. These unique structures exert broad influence over biologically important processes and can modulate cell survival and human health. In fact, mutations, hyper-stability, and dissociation of GQs are implicated in neurodegenerative disease, mental retardation, premature-aging conditions, and various cancers. As such, GQs are novel drug targets. GQ-targeting therapeutics are developed to influence the folding and genetic interactions of GQs that are implicated in diseased states. To do so requires a greater understanding of GQ structure and dynamics and molecular dynamics (MD) simulations are well suited to provide these fundamental insights. Previous MD simulations of GQs have provided limited information due to inaccuracies in their models, namely the nonpolarizable nature of their force fields (FFs). The cutting-edge Drude polarizable FF models electronic degrees of freedom, allowing charge distribution to change in response to its environment. This is an important component for modeling ion-ion and ion-DNA interactions and can influence the overall stability of GQ structures. The work herein employs the Drude polarizable FF to 1) describe the role of electronic structure on the dynamics and folded stability of GQs, 2) determine the impact of ion interaction on GQ stability, and 3) characterize the role of G-hairpin motifs in GQ intermediates. Such fundamental investigations will help clarify GQs role in healthy and diseased states and transform our understanding of noncanonical DNA, improving human health, therapeutic design, and fundamental science.

All viruses are dependent on interactions with host cellular proteins to perpetuate their lifecycles. The genus Flavivirus, one of the four genera that comprise the family Flaviviridae, contains members that cause significant morbidity and mortality worldwide. Among the mosquito-transmitted flaviviruses, West Nile Virus (WNV), dengue (DENV), and Zika virus (ZIKV) have emerged in the Western hemisphere, which now joins Southeastern Asia and Africa as an endemic region. There are no effective antiviral therapies for treating individuals with flavivirus infections, and only a few flavivirus vaccines are available for humans. Current knowledge about the complex mechanisms that differentially regulate flavivirus positive and negative strand RNA synthesis during the various stages of the replication cycle is incomplete. Previous work identified a multi-contact site interaction between the cell protein eEF1A and the conserved flavivirus 3′(+)SL RNA as well as an interaction between the cell protein TIAR and the viral 3′(-)SL RNA. Both of these interactions have been shown to affect viral replication. The formation of TIAR-RNA complexes was studied using a number of biochemical and analytical chemistry techniques. Further studies were performed to examine TIAR complex formation in the context of the 3′(-) WNV SL. Finally, a structural analysis of EF1A1 was preformed using a deposited cryo-EM structure. Ultimately, elucidating the structures and functions of these host cellular protein-viral RNA complexes is essential for fully understanding flavivirus replication cycles and for the future rational design of broad spectrum anti-flavivirus therapies.

Biomarker measurements in blood are promising for various clinical applications including early detection of disease and monitoring response to treatment. However, a major challenge with methods for measuring biomarkers in blood is lack of analytical sensitivity. While biomarker levels are typically elevated in the affected tissue, once they enter the bloodstream, they become diluted and decrease in concentration. Many clinically-relevant biomarkers are present at sub pg/mL concentrations in the blood, rendering them undetectable using conventional techniques. To overcome this limitation, ultra-sensitive digital detection of protein biomarkers using Single Molecule Arrays (Simoa) was previously developed. Simoa is 1000X more sensitive than the conventional enzyme-linked immunosorbent assay (ELISA) and has detection limits in the femtomolar range. To further improve the sensitivity of Simoa and enable detection of new protein biomarkers, we developed a method for single molecule protein detection with attomolar sensitivity using droplet digital ELISA (Cohen et al., ACS Nano 2020). We also evaluated how different antibody biotinylation approaches can improve the sensitivity of Simoa immunoassays (Cohen et al., Bioconjugate Chemistry, 2018). To expand the capabilities of Simoa for measuring different types of biomarkers, we developed a new method for microRNA detection (Cohen et al., Nucleic Acids Research 2017) and rat cytokine detection in blood (Cohen et al., Journal of Immunological Methods, 2017). We also applied Simoa assays to various clinical applications. We assessed the impact of clinical sample handling and processing on ultra-low level measurements of plasma cytokines (Cohen et al., Clinical Biochemistry, 2019). We also developed a blood biomarker panel for detecting early-stage breast cancer and discovered biomarkers for monitoring response to immunotherapy in lung cancer patients (Keegan et al., Journal for ImmunoTherapy of Cancer, 2020). Taken together, this thesis work has shown that single-molecule detection of biomarkers in blood is promising for various clinical applications.

Electrochemical aptamer-based (E-AB) sensors are a class of biosensors that incorporate single-stranded DNA or RNA oligonucleotides as recognition elements. Signal transduction for this class of sensor relies on a conformation change of these aptamers in the presence of target that alters the collisional frequency of a 3’-redox reporter. As such, electrochemical sensors that employ nucleic acids as recognition elements, require an understanding of the biophysics of the surface-bound nucleic acid to aid in the rational design of sensors and control over the analytical performance. The work in this dissertation aims to understand how nucleic acid structure and flexibility affect the resulting electrochemical signal observed when either change. This dissertation describes, the extensively characterized electrochemical response of short (

Cancer is a complex, multi-faceted, and varied disease that comes with a substantial economic and health burden. Due to this immense burden and its increasing prevalence, diagnostic tools capable of detecting cancer and aiding treatment, especially at an early stage, have grown into a major area of interest for researchers and clinicians alike. There has especially been a growth in interest in liquid biopsies, which are assays that utilize biomarkers found in typical body fluids like blood and urine in order to diagnose cancer in the same way tissue biopsies normally do, but without the need for invasive procedures. Although cancer biomarkers come in many different forms, two in particular that have received the attention of researchers for liquid biopsies are circulating tumor DNAs and microRNAs due to their stability in body fluids and differential expression in healthy individuals versus cancer patients. Several technologies have arisen and established themselves as gold standards for detecting and analyzing nucleic acid biomarkers, in particular, Polymerase Chain Reaction (PCR) and Next Generation Sequencing (NGS). While these techniques possess excellent sensitivity and throughput, respectively, they have drawbacks that include false negatives from amplification bias, primer mismatching causing false positives for short sequences like those of miRNAs, and laborious preparation for both techniques and extensive analysis for NGS. Our group recently developed a kinetic fingerprinting-based detection method that counts single target molecules directly from sample with ultra-high specificity. This amplification-free approach, termed single-molecule recognition through equilibrium Poisson sampling (SiMREPS), immobilizes potential target molecules on a microscope slide surface and fingerprints them via binding and dissociation patterns of a freely diffusing fluorescent probe. This approach, however, had an upper limit on analysis speed as a higher probe concentration is necessary to accelerate binding speed and the associated background worsens the signal-to-noise ratio. This dissertation focuses on the development of a kinetics-based biosensor design, termed iSiMREPS, which incorporates intramolecular Förster Resonance Energy Transfer (FRET) to rapidly identify nucleic acid targets through conformational changes in the biosensor. Chapter 2 highlights the biosensor design including the three component strands that comprise it called the anchor, capture and query strands and the development of a standardized design capable of rapidly alternating between two conformations to generate a target-identifying FRET signal pattern. In this chapter, I detail the optimizations necessary for the biosensor to perform effectively including capture probe modifications, optimization of the query probe and a competing sequence on the anchor for conformational flexibility and desirable kinetic behavior, and the introduction of the denaturant formamide to provide faster signal generation. Chapter 3 focuses on developing a full assay with the biosensor and details my optimizations to the imaging and slide preparation protocol, maximizing the effectiveness of formamide, improving sensitivity through toehold mediated strand displacement of target-unbound biosensors, and establishing limits of detection for a microRNA target as well a DNA target performed alongside a collaborator. Chapter 4 addresses multi-target detection and shows my development and refinement of biosensors for additional targets, the establishment of design principles and guidelines for generalized target detection, and the development of a multi-target well setup for use in panel assays. Combined, this thesis has developed a proof-of-concept and case for using a biosensor-based assay for liquid biopsy detection, eventually in a spatially addressable microarray format.

Nucleic acids are key macromolecules of living organisms transferring genetic inheritance from one generation to the next. From how a living individual is created to how it interacts with external factors, all and all, can be found in the nucleic acid sequences inside every single cell of every organism. Therefore, the analysis of nucleic acids sequences is a critical capability for cancer and pathogen diagnoses, genotyping, and disease monitoring. To date, numerous methods have been used to detect both characterised and uncharacterised mutations and sequence variations. However, the detection of low amounts of mutant genes in the presence of high levels of wildtype sequences is still a challenge, and existing technologies has room for improvement. The classical approaches for nucleic acid detection and analyses mainly include DNA sequencing and the polymerase chain reaction (PCR). Although these methods with high analytical performance and reliability have facilitated the interrogation of the nucleic acids, some key obstacles such as the need of labelling, high costs for routine clinical use, slow turnaround time for giving results, the complexity of operation, and the inability to dually detect the genetic mutation in one step have limited their applications. To avoid drawbacks of the traditional approaches, a number of chip-based methods leverage electrochemical readouts or microfluidics to identify nucleic acids. However, there is still an unmet need for a less complex, rapid, low-cost, sensitive and accurate method to enable nucleic acid analysis even in resource-poor settings. The overall objective of this PhD thesis is to develop simple, inexpensive and accurate platforms for nucleic acid evaluation. To achieve the aforementioned goal, the first attempt was to develop a lab-on-a-chip platform for cancer diagnosis by detection of circulating tumor nucleic acids (ctNAs) in plasma samples of cancer patients. ctNAs are fragmented DNA released from cancerous cells and tumours into the bloodstream of patients with cancer. Tumour-specific (epi-)genetic alterations in ctNAs are assumed to reflect tumour burden and could be of high value for cancer diagnosis, prognosis, and management. In the first part of the thesis, I developed a new electrochemical assay for the detection of FGFR2:FAM76A fusion gene in ctNAs extracted from ovarian cancer patients. The assay was based on the high electrocatalytic activity of a new class of superparamagnetic graphene-loaded iron oxide nanoparticles. Electrochemical detection demonstrated a limit of detection (LOD) as low as 1.0 fM, high specificity and excellent reproducibility. In the second part of the thesis, I designed and developed a real-time and quantitative PCR system for microbial source tracking (MST) in water samples. MST is a DNA-based technology that enables water-quality managers to identify sources of faecal pollution in environmental waters. Most of the MST methodologies typically require specialized and costly equipment, elaborated and time-consuming operations as well as trained personnel. Here, a simple, low-cost, and sensitive platform was implemented on a microfluidic array chip. The array was successfully used for the real-time PCR-based multiplex detection of three human-associated MST markers (H8, Gen bac III, UidA). The PCR mixture was loaded into an array of channels in a single step utilising capillary filling without the need for liquid handling instruments. The array was then integrated with our custom-made thermal cycling and optical detection system. By employing the fabricated platform, the LOD of 71.8 DNA copies/μL was achieved for Gen bac III sequence. In summary, we introduced a sensitive, simple and economical real-time and quantitative PCR system for MST in water samples. In a further study, I investigated how a nucleic acid amplification setup can be miniaturised. To reach this goal, I utilised liquid marbles as an ideal biochemical microreactors for targeted amplification of the NAs. Liquid marbles are formed by encapsulating microscale volume of liquid with a thin layer of hydrophobic particles. Miniaturization of the nucleic acids (NAs) amplification process inside a liquid droplet provides several advantages upon routine methods, such as reducing reagents consumption and contamination possibility, easy handling of liquids, eliminating the usage of disposable plastic consumables for carrying out biochemical reactions. However, one of the major concerns in liquid marble applications is the high rate of evaporation through the porous walls during the thermal cycling step. To eliminate the evaporation, I used core-shell beads synthesized from a composite liquid marble as a NAs amplification micro reactor comprising two non-miscible liquid droplets forming a spherical shape and a coating of hydrophobic powder. The shell liquid was then polymerised into a solid after exposure to blue light, converting the liquid marble into a core-shell bead. Fabricated core-shell beads were extended to explore their potential as a versatile bioreactor for phylogrouping of the E.coli strains. In general, this platform provided easy manipulation and storage of sample, elimination of the evaporation, and sample protection from possible external contamination. Moreover, this simple and effective method presented a sensitive and inexpensive way to track NAs. In conclusion, this research endeavour presents a step forward towards the adaption of the selected group of tools and technologies, for the development of assays that can be applied as powerful alternatives to conventional tools used in molecular diagnostic. These technologies have the potential to revolutionise the NAs-based diagnostic approaches, by providing sensitive, rapid, accurate, and inexpensive platforms for point of care devices and in-field tests.

The ETS family of transcription factors bind to site-specific DNA via DNA-binding domains called the ETS domains. The ETS domains are structurally homologous but divergent in primary sequences. PU.1 is an essential transcription factor and its biological activity is primarily controlled by up- and down-regulation of its expression. Aside from down-regulated expression, only a few inhibitory mechanisms are known for PU.1. The most understood one involves PU.1 forming a heterodimer with other protein partners, such as GATA-1. However, unlike auto-inhibited ETS-family members whose activity is regulated by autoinhibitory elements that reduce the net affinity of binding to specific DNA, PU.1 has no such regulatory mechanism at the protein-DNA level. We report here that PU.1, unlike its auto-inhibited paralog Ets-1, forms a 2:1 complex with site-specific DNA (>10 bp) in a negatively cooperative manner. We also detected potential interface (193DKDK196) of the PU.1 dimer by using heteronuclear single quantum correlation (HSQC) NMR. Self-titration of PU.1 is a negative feedback mechanism at the protein-DNA level. Following these findings, our group found the presence of the IDRs flanking the ETS domain does not change the DNA binding modes of the PU.1 ETS domain, yet the PEST domain modifies DNA recognition by the ETS domain through changing DNA binding affinities. We successfully assigned ~90% or more backbone amide resonances in the 1H-15N HSQC spectra of hPU.1 constructs with and without IDRs, in the absence and presence (1:1 complex) of DNA. Using the fully assigned HSQC spectra, we studied fast (ps to ns) time scale internal dynamics of PU.1 protein. Spin relaxation rates and heteronuclear 1H{15N}-NOE were acquired for the hPU.1 proteins with and without DNA by NMR. We demonstrated that the PEST domain remains disordered but becomes more dynamic upon specific DNA binding. In terms of DNA recognition, the presence of the PEST domain increases the affinity of 1:1 complex of the ETS domain with cognate DNA, without perturbing the structure or changing the fast time scale backbone motions of the ETS domain.

Nucleic acid X-ray crystallography faces crystallization as well as phasing challenge. The Phasing problem could be rationally solved by incorporation of selenium into the DNA or RNA oligonucleotides, while the crystallization of nucleic acid is still challenging. To address these challenges without structural perturbation, we decided to explore the atom-specific incorporation and place a selenium atom on the 2’-beta position to control B-form DNA formation during crystallization process. Herein we report the first synthesis of the β-2’-MeSe-thymidine (2’SeT) phosphoramidite and Se-DNAs. We found that the Se-DNAs and Se-DNA-protein complexes formed crystals most of time with higher quality and diffraction resolution than the non-modified ones. Surprisingly, the Se-DNA can form crystals up to 600 microns in size, which were frequently hundreds of times larger in volume than the corresponding native. Moreover, we discovered that the high-quality Se-DNA crystals offered the diffraction resolution up to 1.15 Å and the Se-derivatized structure was virtually identical to the native one. In the meanwhile, we synthesized the β-2’-MeSe-cytidine (2’SeC) phosphoramidite and its DNA oligos. To our surprise, the selenium modification greatly facilitate crystallization when the modified cytidine was placed at the terminals of the oligo. The crystal of the Se-DNA formed within a few hours, where the corresponding native crystalized over 1 week. Furthermore, we describe the first synthesis of 2’-MeSe-arabinouridine (2’SeU) phosphoramidite and its DNAs to investigate the structure of DNAs containing the uracil. The Se-derivatized DNA X-ray crystal structure (1.25 Å) was virtually identical to the native one. Interestingly, during the Se incorporation step, we also found that the MeSe group attack the C-4 of the uracil generating a oxazolinyl-selenolester when the N-3 was protected with tert-butyloxycarbonyl (Boc) group which may activate the C-4 position through a n-π* interaction. Besides the structure studies, we also synthesized the 5-phenylselenium and 5-phenyltelenium modified deoxyuridine and incorporated them into DNA oligos to investigate their charge transport properties. The conductance and current-voltage (I-V) characteristics measurement indicates that the Te modification more effectively manipulate the electronic structure of the DNA compared to the Se modification and the corresponding native.

The development of biosensors for the detection of biological molecules has been subject to an intensive research. Nucleic acid detection of both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) has been the most accurate technique used to date for diseases diagnosis and research purposes. Here, we are presenting a silicon-based nanobiosensor for label-free detection of nucleic acids using aptamers-adsorbed nanostructures such as silicon nanowires (SiNWs). The detector consists of nano-structured materials and will be placed within, a microfluidic embodiment. The application of nano-photonics principles allows for detecting changes in absorption when there is a minute change in the surroundings of the nanostructured material. Using previously developed protocols to fabricate the nanostructured materials with various geometries, and functionalize them with nucleic acid aptamers, we investigated and developed novel structures of solid and hollow silicon nanowires with light absorption that is sensitive to changes in the surrounding molecules. The nanostructured material is first coated by immobilized aptamer, and absorption is measured. A change in absorption occurs when the target nucleic acid fragments hybridize with its complementary immobilized aptamer on the nanostructured material. The nano-biosensor presented here is a novel device with applications in disease diagnostic as well as drug discoveries, as it is an accurate, affordable, rapid and easy-to-use in both clinical and research settings.

The further study of the fundamental physical properties of nucleic acids, can have far reaching impacts. These highly dynamic biological macromolecules can be difficult to study as their behavior can be dictated by processes that act on timescales that range over several orders of magnitude. Certain experiments of nucleic acids in solution can scratch the surface of the sub millisecond regime, but experiments that probe processes much faster than that can become difficult, and often do not contain significant atomistic detail, with the notable exception of site-specific liquid-state NMR. From the nanosecond to microsecond, biological macromolecules can be readily studied by simulation. Through the use of molecular dynamics I've studied an exciting feature of nucleic acids, the Hoogsteen base pair. In recent years support for its biological relevance has increased. Understanding the mechanics of how this base pair forms and it's energetic comparison to the Watson-Crick base pair, can lead to developing a greater understanding of it's role in biology. Often comparing to experiments I have seen a stark contrast in the abilities of DNA and RNA to maintain this base pair. In an attempt to explain this difference, I have seen how cooperative shifts in the sugar puckers of DNA, that are necessary for the Hoogsteen base pair to form, are unobtainable in more rigid RNA sugars. I have also been able to observe how the dynamic equilibrium that exists between Watson-Crick and Hoogsteen base pairs within DNA can be influenced by the binding of intercalating drugs, such as echinomycin. Finally, I have combined previous computational techniques to develop a novel way of obtaining time correlation functions from accelerated milestoning techniques, with potential application to biological systems such as the Watson-Crick Hoogsteen base pair transition.

Scaffolded DNA origami uses DNA as building blocks to create 3D nanostructures. Dynamic 3D DNA origami nanostructures have strong potential in biophysical experiments and applications. This includes measuring biomolecular forces, particularly on the nanoscale. Current biophysical methods lack the ability and/or specificity to measure on such a scale, in addition to being challenging and expensive. Dynamic DNA origami can address such a problem by providing a malleable material to accommodate shape and function on the 10-100nm length scale. The major goal of this work is to leverage DNA origami to develop tools to study biophysical properties of the biomolecular complexes.Nucleosomes, consisting of genomic DNA wrapped around a protein core, assemble into higher orders of chromatin structure to compact DNA. Tools to probe site-specific chromatin structure and dynamics at the 10-100nm length scale (relevant for gene regulation) and to apply tensile or compressive forces at targeted sites could greatly improve insight into how chromatin structural dynamics regulate DNA accessibility and processing. We designed, constructed, and implemented a nanocaliper via DNA origami. Our nanocalipers are hinge-like joints that consist of two 70nm rigid arms, each made up of bundled DNA helices, connected along one edge by single stranded DNA.For proof-of-concept, we first studied a single nucleosome by binding the two nucleosomal DNA ends to the ends of nanocaliper arms. Here, the caliper angle reports the nucleosome end-to-end distance. Moreover, the caliper angle is sensitive to nucleosome stability as a function of NaCl concentration. We demonstrated the nanocaliper can detect nucleosome conformational changes via transcription activator Gal4-VP16 binding. The caliper also significantly increases the probability of Gal4-VP16 occupancy by applying a tension to partially unwrap the nucleosome. This suggests that our DNA nanocalipers can report biologically relevant conformational changes and manipulate nucleosomes to test and engineer their function.We developed a model that accurately describes our nucleosome-nanocaliper assemblies as concomitantly (simultaneous but independent) unwrapping, further validated by hexasome-nanocaliper measurements. This model demonstrates nucleosome unwrapping is sensitive to the caliper’s applied force, motivating a design that applies tunable tensile/compressive forces. We created a new nanocaliper with tunable stiffness and equilibrium angle, for example, to incorporate a biomolecule and then apply a tensile/compressive force.This research provides a foundation of future mesoscale studies of nucleosome arrays and chromatin structural dynamics. Broader impacts include these tools directly monitoring or manipulating local chromatin structure and dynamics in single living cells.

The ongoing battle between bacteria and phage has influenced the evolution of complex defense pathways. In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated (Cas) proteins form the basis for RNA-guided adaptive immunity and antiviral defense. The most streamlined CRISPR systems contain the class 2 interference cas genes, which encode a single polypeptide capable of executing all the functions of target search and destruction. This work describes the mechanisms of target recognition and cutting by the class 2 DNA-targeting nucleases, Cas9 and Cas12a (formerly Cpf1), with an emphasis on their biotechnological applications.The ability to program Cas9 or Cas12a to target and cut any DNA sequence by simply changing its guide RNA sequence has revolutionized genome editing. Although Cas9 and Cas12a are both RNA-guided DNA endonucleases, these proteins have little sequence or structural similarity, and several functional differences highlight the evolutionary diversification of CRISPR interference proteins. In particular, Cas9 contains two nuclease domains called HNH and RuvC that cut each strand of the dsDNA substrate to generate a blunt end. The finding that Cas9 undergoes multiple conformational states in the steps leading up to DNA cleavage underscored the importance of conformational control. Using biochemical and single-molecule approaches, we describe a conformational checkpoint that governs activation of Cas9 and uncover a region within the protein responsible for sensing target mismatches that can be further engineered to enhance cleavage fidelity. Unlike Cas9, Cas12a cuts dsDNA with a single RuvC nuclease domain, leaving a staggered end. We find that the RuvC is active only on single-stranded DNA (ssDNA), highlighting the requirement for dsDNA unwinding to facilitate cutting of both strands of the dsDNA substrate. Furthermore, we reveal that once Cas12a binds to a complementary ss- or dsDNA sequence, the RuvC nuclease non-specifically degrades any ssDNA molecule in trans. In the presence of a reporter molecule, this trans-cleavage activity by Cas12a is repurposed as a versatile DNA detection tool and has attomolar sensitivity when coupled with isothermal amplification. Together, the mechanistic framework of Cas9 and Cas12a provides a foundation for engineering fidelity and developing novel DNA detection technologies.

Nucleic acids are biomolecules found in all living things in great abundance and are most commonly found in cells. The two principal nucleic acids are DNA and RNA. DNA’s primary function is to encode genetic information for amino acid sequences that make up proteins that are important in cell functions. RNA has various roles that include regulation and expression of genes.MicroRNAs (miRNAs) are small RNAs, which range between 20 to 25 nucleotides, that bind to mRNA targets and regulate their translation. Circulating miRNAs are bound to various carriers, including nucleoprotein complexes, lipoproteins, exosomes and microvesicles. The carriers protect the miRNAs from degradation and are able to provide transportation to malignant cells where miRNAs may contribute to tumor progression and metastasis. Functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. Aptamers are short, single stranded oligonucleotides, usually around 30 to 50 nucleotides in length that bind to their targets with high affinity and specificity and are comparable to antibodies. Aptamers are discovered through a process known as systematic evolution of ligands by exponential enrichment (SELEX) and necessitates recovery of the ssDNA. An efficient recovery of ssDNA aptamers in SELEX is required to develop high affinity DNA aptamers capable of selectively binding a target molecule. The most common extraction methods employ either silica columns or TRIzol reagent, but these approaches afford low recovery for small RNAs, possibly due to their short strand lengths. We fabricated the titanium dioxide nanofibers using electrospinning to facilitate DNA and miRNA extraction and developed the optimal buffer conditions to improve DNA and miRNA recovery from biological buffers, cell lysate, and serum. Improving the recovery of nucleic acids (NA) could result from the strong coordination between TiO2 and NA’s phosphate backbone. Our results demonstrated that TiO2 nanofibers can work as a valuable tool for extraction of NA from complex biological matrices with high recovery.

Nucleic acids are complex macromolecules that are susceptible to both endogenous and exogenous damage. This study explored damage resulting from interactions with environmental nucleophilic toxins, such as a variety of diols and amines found in industry. These nucleophiles can react with electrophilic groups, such as 5-formylcytosine. 5-formylcytosine is an oxidation product of the epigenetic base 5-methylcytosine. It is typically removed by thymine DNA glycosylase (TDG) but is known to accumulate in the genome, making the formyl group susceptible to attack. In this study we used GC/MS and ESI-MS to show that DNA lesions from the nucleophilic addition reaction of toxins and 5-formylcytosine can be formed under physiological conditions. In addition, this formation showed a pH dependency, with lower pHs showing more product formation. Studies with a lesion formed from the reaction of 1,3-propane diol and 5-formylcytosine showed that the lesion has little effect on the conformation of the DNA duplex. UV thermal denaturation studies showed that at a glance the lesion also has little effect on the stability of the DNA duplex, however, more extensive studies revealed a slight destabilization effect due to the lesion. Enzymatic studies showed that the presence of one lesion does not have a significant effect on the ability of DNA polymerase to efficiently complete DNA replication with high fidelity, but when the lesion was incorrectly base paired, the extension reactions resulted in deletion products or a halt in replication. Addition of a second tandem lesion to the template resulted in a decrease in fidelity, while continuing to give deletion products and replication stops in the presence of mismatched base pairs. This is particularly significant, indicating the potential for the lesion to be mutagenic or even cytotoxic. Lesions formed from other environmental toxins could be even more damaging, making them well worth future investigation.

Biological systems are governed by molecular interactions. At the core of all biological systems is the processing of nucleic acids, and preserving the fidelity of the genome is crucial to those systems. This manuscript follows different biological systems interrogated by nuclear magnetic resonance: the structural impact of single ribonucleotide damage in DNA, the formation of oligomeric protein-DNA complexes of an ETS family transcription factor, how methylation affects the charge state of the amino acid arginine and how the base pair opening rates of an oligonucleotide is affected by inosine.

Biochemical properties of ribonucleic acids enable their instrumentality in physiological functions. RNA sequence serves as the intermediary of the genetic code, while secondary and tertiary structures are responsible for RNA’s involvement in regulation and catalysis. As such, discovery and biochemical characterization of RNAs promise to illuminate the relationship between an RNA’s sequence, structure, and functions, thus provide insights into their biological significance. Efforts in functional RNA discovery often turn to in vitro selection or SELEX (systematic evolution of ligand by exponential amplification) to identify RNAs of a functional trait of interest from a diverse pool of sequences. Selected RNA species are subsequently sequenced and characterized for binding affinity or catalytic activity. Given the size and diversity of the enriched pool at the end of a selection, elucidation of RNA sequence and structure remains a labor-intensive process. The work presented here focuses on massive parallel identification and characterization of functional RNAs emerged from in vitro selections through a bioinformatics pipeline known as Apta-Seq. Enriched sequences from multiple selections were examined via high-throughput sequencing coupled with selective 2´-hydroxyl acylation analyzed by primer extension (SHAPE). These sequences are the output of in vitro selections of RNA aptamers for cyclic-nucleotide monophosphate from genomic DNA libraries. High-throughput analyses of sequences emerged from genomic in vitro selections leads to the discovery of an abundant aptamer with affinity and selectivity towards 3´, 5´-cyclic guanosine monophosphate. Independent structure probing experiments performed on this aptamer produced a binding constant at 5.9 µM and identified key nucleotide residues involved in binding of the ligand. These results established correlation between high-throughput SHAPE data and in-line probing experiments, thus demonstrating Apta-Seq as a sensitive method to massive parallel investigate RNA sequence identity and secondary structure. Moreover, selection of multi-turnover ribozymes of strain-promoted azide-alkyne catalysis was explored in this work. However, the unsatisfactory output of the selection encourages revision of the selection strategy. Nonetheless, the progress of the selection has the potential to address the challenge of RNA in vivo imaging.

The genetic code contains all the information needed to execute every biological task in cells. Although this information is encoded in DNA, proteins are the primary players that execute the biological functions. Understanding how DNA is recognized and regulated by cellular proteins is crucial to gain a deeper understanding of cell biology. Recent advances in mass spectrometry instrumentation allow the investigation of large portions of the proteome simultaneously, making it an attractive technique to examine DNA-Protein interactions. In this thesis, we aimed to develop a quantitative proteomic approach to identify novel nucleic acid-binding proteins. In chapters 2 and 3, we developed and applied a quantitative proteomic method to identify proteins that recognize non-canonical G-quadruplex (G4) DNA. G4s are important biological players due to their genomic locations and their roles in functional biology. We investigated the interaction proteomes of three unique G4 structures. Our technique facilitated the identification of 84 proteins that preferentially bind to G4, including NSUN2 and SLIRP. Upon further investigation, we demonstrate that NSUN2 is a selective binder of G4 structures derived from the promoter of cMYC and cKIT genes, but not that from the human telomere. In chapter 3, we characterized the binding of SLIRP to G4 DNA. We demonstrated that SLIRP can selectively bind all three G4 DNA patterns with low nanomolar binding affinity; the protein, however, exhibited significantly lower binding affinity to single-stranded DNA. Using site-directed mutagenesis, we found that L62 and R24/R25 in the RNA recognition motif of SLIRP are key residues for G4 recognition and binding. In chapter 4, we extended our technique to identify the proteins that interact with two tandem DNA lesions, cdA and cdG. We uncovered 33 proteins can bind to duplex DNA harboring a site-specifically incorporated cdA and/or cdG lesions over their unmodified counterparts. We investigated further one of the putative cdA- and cdG-binding proteins, CDKN2AIP. We knocked out the CDKN2AIP gene and discovered that CDKN2AIP-/- cells displayed significantly poorer survival than wild-type cells when challenged with DNA damaging agents that can induce cdA and cdG lesions, but not those agents that induce other types of DNA lesions.