In alkaline conditions, a complex called M-DNA is formed between a divalent metal ion, cobalt, nickel or zinc, and duplex DNA. The rate of formation and stability of M-DNA is dependent on many factors, including but not limited to temperature, pH, DNA sequence, and metal or DNA concentrations. It has been hypothesized that the divalent metal ions intercalate into the helix and replace the imino protons involved in the hydrogen bonding of both G-C and A-T base pairs. The complex is thought to have a double helical structure that is similar to B-DNA. The presence of the divalent metal ions and a more compact structure may contribute to M-DNAs remarkable ability to behave as a molecular wire. Because M-DNA is so similar to B-DNA, it adheres to the same rules with regards to self-assembly. The ability of DNA to self-assemble and the electronic conduction of M-DNA are ideal properties for nanotechnology of the future. M-DNA may eventually be used to detect the presence of biologically important small molecules and DNA binding proteins that block the flow of electrons. However, before M-DNA will be widely accepted, it is necessary to obtain an accurate 3-dimensional structure by X-ray crystallography and modelling. <p> In this work interactions between divalent cobalt, nickel or zinc with duplex DNA were studied using two different experimental methods; namely, X-ray crystallography and extended X-ray absorption fine structure spectroscopy. First, crystals of the sequence d[GA(5FU)(5FU)AA(5FU)C] and d[CG(5FU)G(5FU)GCACACG] complexed with divalent metals were grown in M-DNA favouring conditions. Both of the sequences gave crystals that provided diffraction data that were analyzed by molecular replacement using B-DNA models. Unfortunately, the quality of the diffraction was not high enough with either sequence to locate metal binding or to determine a model for M-DNA. Second, X-ray absorption spectroscopy data were analyzed for the Ni2+ K-edge of both Ni2+ M and B-DNA. Several differences between the M and the B-DNA data were noticed and some final bond distances were established.

<p>Nucleic acids are subjected to many different mechanical loadings inside. These loadings could cause large deformations and conformational changes to these molecules. This is why the mechanical properties of nucleic acids are so important to their functions. Here we use a newly designed and built high-performance AFM force spectrometer, supplemented with molecular dynamics simulations and NMR spectroscopy to investigate the relationship between mechanical properties and structure of different nucleic acids.</p><p>To test the mechanical properties of nucleic acids, we successfully designed and purpose-built a single molecule puller, an instrument to physically stretch single molecules, at a fraction of the cost of a commercial AFM instrument. This instrument has similar force noise to hybrid instruments, while also exhibiting significantly lower drift, on the order of five times lower. This instrument allows the measurement of subtle transitions as a molecule is stretched. With the addition of a lock-in amplifier, we possibly could obtain better force resolution, the order of femtonewtons. </p><p>We find that helical structure does indeed have an effect on the mechanical properties of double-stranded DNA. As the A-form double helix has a shorter, wider structure compared to the B-form helix, its force spectra exhibit a shorter initial length before the overstretching force plateau, compared to B-form DNA. Contrarily, the Z-form double helix has a narrower, more extended helical structure than B-form DNA, and we see this fact manifest in the force spectra of Z-DNA, which has a longer initial length before the overstretching force plateau. Also, interestingly, we find that neither A, nor Z-DNA force spectra display the second melting force plateau. Indicating this plateau is not necessarily cause by melting of strands apart, but rather a feature of B-DNA. </p><p>To better understand the forces that stabilized these different structures, specifically base stacking, we also mechanically characterize different single-stranded helical polynucleotides using AFM based force spectroscopy. We expand on previous studies by confirming that single helical polynucleotides undergo a force transition at a force of ~20 pN as they are uncoiled, and also demonstrating, that when stretched beyond this force transition, the molecules behave differently depending on base sequence and backbone sugar. Specifically, the force spectra of poly-adenylic acid possess a linear force region, which persists to ~300 pN, after the force plateau. We also observe that poly-deoxyadenylic acid is comparatively stiffer than other polynucleotides after undergoing two force transitions. By supplementing our force spectroscopic data with MD simulations and NMR spectroscopy, we find that base stacking in adenine is quite strong, persisting above 100 pN. We find that initial helical structure, which is defined by base stacking and backbone sugar, guides the stretching pathway of the polynucleotides. This finding can possibly be extrapolated to the elasticity of double-stranded DNA.</p> / Dissertation

Nucleic acids are a uniquely flexible and multi-faceted class of molecules that fulfill fundamental and defining tasks such as replication and determination of heritable characteristics in every living organism. From the microscopic to the gigantic, from the most primitive to the most complex, life has been both molded and served by nucleic acids. Nucleic acid circuits straddle the realm of nature and technology. The elegance of interaction between nucleic acid molecules invites us to gain a deeper understanding of the naturally occurring systems they compose and to apply our ingenuity and foresight toward developing ever more complex synthetic systems. Nature has provided these basic building blocks, which we can now arrange – and augment – for the purpose of creating molecular-level machinery. Here we describe some ways in which we have rationally harnessed nucleic acids. In preparation for outbreaks of novel and deadly avian influenza viruses, we used quantitative polymerase chain reaction (qPCR) to track the number of flu virus particles surviving in the presence of potential antiviral drugs. We engineered tunable on/off switches that can be used to evaluate a series of conditions for diagnostic applications or to enable ‘smart’ drugs that sense, analyze, and respond to their microenvironment. We optimized the conditions for, and used, a unique set of guanine-rich DNA sequences called G-quadruplexes, whose enzymatic and structural properties make them prime effector candidates in diagnostic platforms. G-quadruplex folding powers isothermal DNA amplification, and the small organic molecules they bind endow G-quadruplexes with expanded catalytic abilities. We genotyped drug resistance mutations in tuberculosis via visually detectable color changes in the reaction buffer. We developed a paper fluidics assay that employs soluble and bead-immobilized nucleic acids to scan for genes in tuberculosis, and upon detection, to generate a readily observable discoloration on the paper strip. Finally, we probed the boundary of nucleic acid circuitry by attempting to expand its language via the incorporation of unnatural nucleobases into oligonucleotide components of a catalytic hairpin assembly (CHA) circuit. We subsequently evaluated the resilience of the unnatural CHA circuit to contamination by random DNA species, such as may be encountered in clinical samples. / text

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The generation of synthetic oligonucleotides is dependent on an efficient solid-phase synthesis methodology. This thesis evaluates the 2'-O -levulinyl (Lv) and 2'-O-monomethoxytrityl (MMT) ribonucleosides, as possible synthons for RNA and branched RNA synthesis. A key feature of this RNA and bRNA synthesis procedure is their removal while still attached to the solid support and under conditions that prevent isomerization or cleavage of the nascent strands. For the first time, the stability of 3'-5'-internucleotide phosphate triesters (and diesters) adjacent to a ribose 2'-hydroxyl group was determined on a solid support. These studies are not only relevant to the proper assembly of branched and linear RNA species, but also to the stability of an unusual branched RNA species ("RNA X") proposed to form during the pre-mRNA splicing reactions in vitro. These studies are also important to the development of large quantities of native and chemically modified short interfering RNA (siRNA) for animal and human studies. / The 2'-O-Lv and 2'-O-MMT ribonucleoside monomers served as building blocks for the assembly of a series of branched nucleic acid species (bRNA, bDNA, msDNA and hyperbranched or "dendritic" DNA/RNA) with discrete length, base composition and structure. These structures were synthesized via an iterative divergent-growth strategy, which facilitates the regioselective branching, deblocking and chain lengthening steps from a branchpoint core. These structures served as useful materials (bio-probes) as demonstrated by the biological studies performed with E. coli RNaseH and the yeast lariat RNA debranching enzyme (yDBr1). These studies not only led to the identification of novel branched nucleic acid inhibitors of yDBR1 and RNase H, but also provided new insights about the substrate specificity of these important enzymes. / This thesis also describes the synthesis of a new nucleic acid form, the so-called "oxepane nucleic acids" (ONAs), in which the pentofuranose ring of DNA and RNA was replaced with a 7-membered heptose sugar ring. ONA were found to be much more resistant towards nuclease degradation than natural DNA, an important feature if these analogues are to be used in biological media. Furthermore, ONAs exhibited cross-pairing with complementary RNA and were found to elicit E. coli RNaseH mediated degradation of the RNA strand. These finding are significant because oligonucleotide-directed RNase H degradation of the target RNA is a key determinant for the gene-specific inhibitory potency of antisense oligonucleotides. When comparing the rates of RNase H-mediated degradation induced by 5 (furanose), 6 (2'-ene-pyranose) and 7 (oxepane) membered ring oligonucleotides, the following trend was observed: DNA > 2'-ene-pyranose NA > ONA. The implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme, particularly with regard to the required flexibility of the oligonucleotide strands that bind to the RNA target. Hence, ONAs are useful tools for biological studies and provide new insights into the structure/function of natural and alternative genetic systems.

This thesis presents the development of novel methodologies in the template mediated chemical synthesis of lariat and branched nucleic acids. The synthetic branched DNA and RNA may be applicable as probes in the elucidation of the splicing mechanism or as potential therapeutic agents. Furthermore, this body of work describes the novel synthesis of Ru(II) branched DNA as building blocks in the supramolecular assembly of nano-motifs. In general, insight into the utilization of nucleic acids as biological molecules and as nanomaterials is presented at the interface of chemistry and biology. / Chapter 2 delineates the regioselective template directed synthesis of Y-RNA via chemical ligation at the branch point of a 5'-phosphate to a 2'-hydroxyl. The branched molecules resemble lariats as they possess the analogous branched architecture. The oligonucleotide components are synthesized from commercially available phosphoramidite building blocks through automated solid-phase synthesis. A unique template directed method in the synthesis of DNA and RNA lariats is proposed in Chapter 3. The regioselective chemical ligation affords wild-type DNA and RNA formed through assembly of a single oligonucleotide strand. A parallel DNA:RNA hybrid association was observed in the preorganized assembly and extensively characterized. Characterization of the Y-RNA and lariat nucleic acids were carried out through techniques such as thermal denaturation analysis, polyacrylamide gel electrophoresis, enzymatic degradation with the RNA lariat debranching enzyme, alkaline treatment as well as MALDI-TOF mass spectrometry. / The second part of the thesis exploits DNA as a nanomaterial in the convergent solid-phase synthesis of Ru(II)-DNA conjugates as branched building blocks in the assembly of nanostructures. Chapter 4 describes the synthesis of Ru branched DNA, utilizing cis-[(bpy)2Ru(imidazole) 2]2+ moiety as the vertex tethered to parallel DNA covalently through flexible hexamethylene linkers. Complete physical characterization and preliminary hybridization studies are conducted. The Ru-DNA conjugates presented were found to be unstable to the protocols required for synthesis of mixed sequence derivatives. The stability and scope of synthesis of these molecules are further discussed. / As an alternative, Ru-DNA branched complexes of mixed sequences, exhibiting greater stability, were synthesized. The transition metal building blocks of Chapter 5 employ a more rigid branch point, linking two parallel DNA strands through a one methylene spacer to the cis-[Ru(bpy)2 (4,4'-bis(hydroxymethyl)-2,2'-bipyridine)][PF6]2 vertex. Physical characterization and the intrinsic luminescent properties of the transition metal complex were confirmed in both the Ru-branched DNA and hybrized forms. A comparative study of the self-assembly behavior of the Ru-DNA conjugates to that of unmetallated branched DNA was also conducted. Interestingly, results indicate a metal-mediated assembly of almost exclusive formation of one discrete Ru-DNA dimeric cyclic nanostructure, where as unmetallated DNA building blocks produced an array of products. Complete confirmation of these products is presented through PAGE and enzymatic digestions. Finally the synthesis of novel Delta and &Lambda; Ru-branched DNA diastereomers is presented as potential building blocks in the creation of chiral metallo-supramolecular constructs.

The research presented in this thesis introduces design criteria for development of a new type of self-contained optical biosensor. The study begins with evaluation of a dual label, fluorescence resonance energy transfer (FRET) bioassay format, and then goes on to demonstrate a signalling platform that uses an immobilized fluorescent intercalating dye so as to avoid labelling of both the target and probe strands. An extensive survey of FRET pairs that can be used to monitor hybridization events in solution and at solid interfaces was conducted in solution to provide a set of calculated Förster distances for the extrinsic labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethylrhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ) and Iowa Black RQ (IabRQ). FRET parameters using thiazole orange (TO) intercalating dye as a FRET donor for various acceptor dye-labelled DNA conjugates in solution were determined. Limitations associated with quenching mechanisms other than those mediated by FRET motivated the development of a molecular switch that contained intercalating dye. The four binding sites associated with Neutravidin served for assembly of the switch using biotin interactions. One binding site was used to immobilize an unlabelled oligonucleotide probe. The adjacent site was used to immobilize a novel biotinylated TO derivative that could physically reach the probe. On hybridization of the probe with target, the intercalating dye was captured by the hybrid, leading to a change of fluorescence. This reversible signalling mechanism offers a method without nucleic acid labelling to detect nucleic acid association at an interface. A SNP discrimination strategy involving TO and formamide was investigated, and SNP discrimination without the requirement of thermal denaturation was achieved for multiple target lengths, including a 141-base pair PCR amplicon in solution. It was determined that formamide could also provide improvements of signal-to-noise when using thiazole orange to detect hybridization.

The research presented in this thesis introduces design criteria for development of a new type of self-contained optical biosensor. The study begins with evaluation of a dual label, fluorescence resonance energy transfer (FRET) bioassay format, and then goes on to demonstrate a signalling platform that uses an immobilized fluorescent intercalating dye so as to avoid labelling of both the target and probe strands. An extensive survey of FRET pairs that can be used to monitor hybridization events in solution and at solid interfaces was conducted in solution to provide a set of calculated Förster distances for the extrinsic labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethylrhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ) and Iowa Black RQ (IabRQ). FRET parameters using thiazole orange (TO) intercalating dye as a FRET donor for various acceptor dye-labelled DNA conjugates in solution were determined. Limitations associated with quenching mechanisms other than those mediated by FRET motivated the development of a molecular switch that contained intercalating dye. The four binding sites associated with Neutravidin served for assembly of the switch using biotin interactions. One binding site was used to immobilize an unlabelled oligonucleotide probe. The adjacent site was used to immobilize a novel biotinylated TO derivative that could physically reach the probe. On hybridization of the probe with target, the intercalating dye was captured by the hybrid, leading to a change of fluorescence. This reversible signalling mechanism offers a method without nucleic acid labelling to detect nucleic acid association at an interface. A SNP discrimination strategy involving TO and formamide was investigated, and SNP discrimination without the requirement of thermal denaturation was achieved for multiple target lengths, including a 141-base pair PCR amplicon in solution. It was determined that formamide could also provide improvements of signal-to-noise when using thiazole orange to detect hybridization.

Nucleic acids are responsible for the storage of genetic information and directly participate in gene replication, transcription and expression, and thereby the control of nucleic acids leads to the regulation of genetic information flow and gene expression. Meanwhile, many non-coding RNAs are in-volved in signal transduction directly. Moreover, nucleic acid-based therapeutic strategies have been lead to drug candidates and are effective tools in drug discovery and disease study at the molecular level as well as the genetic level. Consequently, the 3D crystal structure study and related functional research on natural and unnatural nucleic acids have become very popular area, expanding their potential appli-cation in medicinal and biological chemistry. Since oxygen, sulfur, selenium and tellurium are in the same elemental family (VIA) in the peri-odic table, we anticipate that oxygen atoms in nucleic acids can most likely be replaced with the other chalcogen atoms without causing significant perturbations. Owing to the special K edge and unique properties of selenium, our lab has completed the chemical and enzymatic synthesis of unnatural nucle- ic acids with selenium substitutions at various positions. The selenium functionality in nucleic acid is es-sential for nucleic acids’ structural determination at the atomic level. Additionally this novel elemental feature (atomic size and electronic nature) provides nucleic acids with unique properties. In addition, the selenium derivatization can facilitate crystal growth. Other chalcogen elements are applicable as well to modify nucleic acid, generating some special biofunctions, like the application of phosphorthioate oligonucleotide in gene therapy. This dissertation will outline the chalcogen elements (especially selenium) modifications of nucleic acids, including syntheses strategies, structure studies and potential therapeutic applications. Our research work here tries to show that (1) Selenium functionality is able to facilitate the crystal structure determination, by both helping solve phase problem and accel-erating crystal growth; (2) Selenium functionality can generate special capability to nucleic acids, like improved base pair fidelity, novel atomic interactions and feasibility to be biological chemistry probe; (3) Selenium derivatized oligonucleotides are extraordinary good candidates for gene therapy discovery, considering its stability under nuclease environment. In general, these atom-specific replacements gen-erate a new paradigm of nucleic acids. INDEX WORDS: Nucleic acid, Selenium, X-ray crystal structure, Biofunction, Therapeutics