Many different experimental approaches were attempted to achieve carbon-carbon bond formation by nucleic acid template-directed reactions and ribozyme catalysis as potential lipid synthesizing machineries in the RNA world. A novel biomimetic condition for decarboxylative Claisen condensation in polyketide biosynthesis was discovered. The reaction of a malonic acid half oxyester with a Nhydroxysuccinmidyl ester forming reagent resulted in self-condensation to provide the corresponding 1,3-acetonedicarboxylic acid diester in the absence of a divalent metal chelator or a coordinating solvent. The decarboxylative Claisen condensation of malonyl adenosine using a poly-U template in solution or with immobilized poly-U was attempted. Various analytical methods demonstrated that malonyl adenosine underwent an exclusive hydrolysis reaction instead of condensation in the given conditions. Similar results were observed for the reaction of malonyl-CoA with acetyl-CoA on poly-U templates. No evidence for the decarboxylative Claisen condensation was observed by a DNA-templated system although a double helical structure of DNA duplex was proven to facilitate a bimolecular reaction by offering a favorable proximity effect. Therefore, it seems that the unsuccessful condensation resulted not from the bad template effect but from the intrinsic properties of the decarboxylative Claisen condensation reaction itself. Two tRNA molecules loaded with a malonic acid were prepared by ligation of truncated tRNAs with malonylated dinucletides. Our initial attempts to probe carbon-carbon bond formation by subjecting malonylated tRNAs to the in vitro translational machinery were not successful. Novel carbon isosteres of α-amino acids are suggested as a potential source of a more stable and reactive carbanion for future experiments. Isoprenoid conjugates of nucleoside 5??-diphosphates, which were proposed as either an initiator nucleotide or substrate molecule for in vitro selection of prenyl-transferase ribozyme were prepared by one step nucleophilic displacement reactions. A random DNA pool was constructed for selection of a ketosynthase ribozyme. A substrate bearing a biotin tag was prepared by one-step conjugation. Hig-tagged T7 RNA polymerase was expressed and purified for a large scale transcription reaction. In vitro transcription of the random DNA pool with a 5??-thiol modified GMP analogue as an initiator nucleotide produced a thiol-modified random RNA library.

In this thesis, we document a comprehensive study of the cation-directed self-assembly of three guanosine derivatives: i) guanosine 5'-monophosphate (5'-GMP), ii) guanosine 5'-thiomonophosphate (5'-GSMP), and iii) 2',3',5'-O-triacetylguanosine (TAG). We discovered that, under the neutral pH condition, Na2(5'-GMP) molecules self-assemble into a right-handed helix structure consisting of alternating all-C2'endo and all-C3'endo planar G-quartets stacking on top of each other with a 30° twist. This self-assembled supramolecular structure uses multiple non-covalent forces (e.g., hydrogen-bond, phosphate-hydroxyl, pi-pi (base-base) stacking, ion-carbonyl, and ion-phosphate) to align individual monomers in a way that resembles RNA and DNA sequences in which covalent bonds are used to link monomers. Na+ ions are located in the channel and surface sites of the G-quadruplex. In contrast, under acidic pH conditions, Na2(5'-GMP) molecules self-assemble into a continuous right-handed helix where guanine bases are hydrogen-bonded in a lock-washer fashion with only C3'-endo monomers. Na+ ions are absent in the channel site due to smaller channel radius and lesser repulsions between phosphate groups (-1 vs. -2 charge under neutral pH) contribute to the stronger stacking mechanism. In Na2(5'-GSMP), a longer phosphate bond compared with Na2(5'-GMP) allows stronger P-O-…Na+…-O-P interactions to occur, thus enhancing self-assembly. Solid-state NMR, FT-IR, powder x-ray diffraction, model building, and calculation showed that Na2(5'-GSMP) forms the same self-assembled structure as Na2(5'-GMP) but with significantly greater tendency. This study proves that single-bond modification can enhance stacking in G self-assemblies, and shows direct evidence that Na+ ions reside at the surface (phosphate) sites. Lastly, using lipophilic TAG, we were able to show for the first time that trivalent lanthanide metal ions can facilitate G-quartet formation. A new mode of metal ion binding in G-quartet structures (i.e., a triple-decker G dodecamer containing a single metal ion in the central G-quartet) is reported. We also report the first 1H and 43Ca NMR characterization of Ca-templated G-quartet formation in a [TAG8-Ca]2+ octamer. / Thesis (Ph.D, Chemistry) -- Queen's University, 2012-06-27 16:53:43.359

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The increasing number of nucleic acid-based therapeutics demonstrates the potential to treat diseases at the genetic level. Although oligonucleotides show clinical potential, challenges remain including nuclease degradation, rapid clearance when administered systemically, low cell permeability, and limited distribution to tissues of interest. This is largely imparted by the polyanionic phosphate backbone, which produces unfavourable electrostatic interactions at cell membranes. As a result, their clinical translation is dependent on delivery technologies that improve stability, facilitate cell entry, and increase target affinity. Spherical nucleic acids (SNAs) consist of radially orienting linear nucleic acids onto a nanoparticle core, conferring them a three-dimensional, spherical architecture. These structures enter cells readily and display distinct properties that are independent of their nanoparticle core. Accordingly, we decided to replace the intrinsically anionic phosphodiester linkage of DNA with a phosphoramidate linkage (P-N), allowing us to incorporate new functionality at the phosphate backbone. With this handle, we inserted cationic and hydrophobically modified functional groups that were compatible with nanoscale architectures, giving rise to new properties relevant in biological contexts. Specifically, amine and guanidinium derivatized functional groups provided SNAs with a ~10-fold increase in cell uptake at early incubation times compared with unmodified SNAs. This demonstrates that we can tune the behaviour of SNAs with phosphate backbone modifications in a highly controlled manner. We hypothesize that the stringent control over location and placement of functional groups within the SNA framework will afford them favourable interactions at cell membranes, not only increasing their cell uptake, but also access to alternative uptake mechanisms and potency as therapeutics. / Thesis / Master of Science (MSc) / Oligonucleotides are short synthetic sequences of DNA or RNA that have the capacity to treat diseases at the genetic level. However, they face challenges such as degradation, low cell uptake, and poor tissue distribution. To overcome this issue, we plan to incorporate chemical modifications at the phosphate backbone of oligonucleotides to make them more stable and facilitate more favourable interactions at cell membranes. Conferring oligonucleotides into a 3D arrangement further enhances their stability and cell uptake relative to linear oligonucleotides. By densely functionalizing them onto a nanoparticle core, we can create spherical nucleic acids (SNAs). We hypothesize that the modifications imparted onto the phosphate backbone of linear oligonucleotides will translate their properties into SNAs. The new properties afforded to the SNAs will provide increased cell uptake, alternative uptake mechanisms, and access to cytosolic and nuclear targets, highlighting their potency and therapeutic potential.

The delivery of nucleic acids at the tissue and cellular levels remains one of the major hurdles in this scientific area. Since nucleic acids are bulky macromolecules and unstable in the presence of nucleases, vehicles are required to compact them into nanosized particles, offer protection from degradation in vivo, and release the therapeutic cargo at the desired location. Polycationic vehicles are good candidates for these purposes since they can be chemically modified to tune the desired properties in nanoparticle formulations. We designed a family of trehalose-oligoethyleneamine copolymers that showed promising plasmid DNA (pDNA) transfection results in the presence of serum proteins. A diazidotrehalose monomer was copolymerized with linear oligoethyleneamines of varying length and containing alkyne end-groups via step-growth Cu(I)-catalyzed azide-alkyne cycloaddition polymerization resulting in a series of trehalose copolymers with a range of secondary amines (from 4 to 6) within the polymer backbone. Upon electrostatic complexation of the polycations and pDNA in aqueous media, nanosized particles were formed, and their sizes and zeta-potentials were characterized via dynamic light scattering (DLS). The glycopolymers were tested for pDNA binding, toxicity, cellular uptake, and transfection efficiency in vitro. Characterization of these polymers revealed a significant influence of minor structural modifications on bioactivity. In general, all of the polymers efficiently bind pDNA at low nitrogen to phosphate (N/P) ratios forming nanoparticles below 100 nm in size and demonstrated cellular uptake and transfection. Polymers comprised of trehalose moieties and four secondary amines in the repeat unit showed the greatest promise in pDNA delivery in vitro. Because of its large hydration volume, we hypothesize that trehalose contributes to particle stabilization in serum. The trehalose-based polymers with four secondary amines (Tr4) were subsequently modified with PEG (5kDa). This modification lead to the development of well-defined polymeric structures with PEG moieties selectively incorporated at the ends of linear trehalose-oligoethyleneamine polycations. The study of the effect of this modification on bioactivity revealed that there were no significant difference in the toxicity profiles within this series of PEGylated and non-PEGylated materials; however, overall results suggest that both modified and unmodified trehalose-oligoethyleneamine copolymers have a great promise for stem cell-based and regenerative therapies. / Ph. D.

Thesis advisor: Jia Niu / Nucleic acid aptamers are promising alternatives to antibodies for a wide array of diagnostic and therapeutic applications. However, state-of-the-art aptamers suffer from poor pharmacokinetics and diversity, limiting their affinity and specificity for many therapeutically relevant targets. The emerging field of glycoscience provides opportunities to improve the utility of aptamers over antibodies. Combining synthetic chemistry with modern molecular biology and polymer science, the synthesis of Xeno Nucleic Acid monomers and chemoenzymatic polymerization via engineered polymerase enzymes allows the production of nucleic acid drugs with superior resistance to endogenous nucleases. The modular structure of nucleic acids provides for the design of sequence defined polymers capable of post-synthetically appending complex synthetic glycans, extending the catalytic geometry of aptamers. Our SELEX inspired FACS based particle display approach allows for high-throughput screening. Additionally, we expect this method has the capability of screening aptamers in human serum. Our synthetic approach utilizes a Sonogashira cross-coupling reaction to install a flexible alkyne to the major groove of 2′-deoxy-2′-fluoro-arabinose uracil base. By incorporated recent advances in nucleic acid synthesis, one-pot nucleobase activation and sugar glycosylation is achieved and bis-oxybenzyl phosphoamidite synthesis can afford gram scale HPLC-free purification of the triphosphates. The FANA C8-alkyl-uridine triphosphate will be incorporated by an engineered Tgo DNA polymerase to allow systematic introduction of alkynyl conjugation handles into a DNA-templated FANA polymer. Subsequent conjugation with azido-modified glycans via the Huisgen coppercatalyzed alkyne-azide cycloaddition (CuAAC) click reaction will generate sequence controlled nucleic acid-carbohydrate hybrid molecules amendable for directed evolution. / Thesis (MS) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Graduation date: 1993

Deoxyribonucleic acid and ribonucleic acid under physiological condition are polyanions composed of heterocyclic bases linked through sugar phosphate backbone. Due to Watson-Crick base pairing, DNA exists in double-helical form between two antiparallel strands of nucleic acid. Different conformations of DNA is possible among which the B-DNA form is considered to be the most common, and it is a right-handed double-helix with base pairs stacked at the center. There are two well-defined grooves termed as major and minor grooves, each has characteristic width and depth. Most of the DNA binding proteins generally approach DNA through the major groove, while small molecules such as drugs, antitumor antibiotics,1 their synthetic analogue,2 carcinogens,3 and the transition metal complexes4 interact with DNA through minor groove. The nucleic acids function in the storage and transfer of genetic information. The function of cell expressions of proteins, synthesis of all bio-materials are directly or indirectly governed by the nucleic acid present in the body. Not only that, the origin of many diseases lie behind the structural modification or alterations in nucleic acids occur beyond our control.5 There are different drugs both natural and synthetic which are important in antibiotic chemotherapy, act against these diseases by interacting with DNA. Now to understand the actual mechanism of many diseases, how drugs interact with DNA and its specificity, binding sites of DNA, we need to develop molecules that modify or interact with biological molecules and such molecules can probe various structural aspects and type of interaction of macromolecular association complexes. One of such probe is the DNA cleaving agent. The potential scope of the utility of these compound is enormous and ranges from the creation of synthetic restriction enzymes for use by molecular biologists to the development of chemotherapeutic agents (Fe(BLM), calicheamicin) that may be effective against a variety of neoplastic diseases. They can also act as a structural probe (e.g. Fe(EDTA)2), drug / protein-DNA footprinting agent and affinity cleaving agent.

No description available.

The design of probes for the selective recognition of biopolymers (nucleic acids and proteins) is a fundamental task for studying, diagnosing, and treating diseases. Traditional methods utilize a single component (small molecule or oligonucleotide) that binds directly to the target biopolymer. However, many biopolymers are unable to be targeted with this approach. The overarching goal of this dissertation is to explore a new, binary approach for designing probes. The binary approach requires two components that cooperatively bind to the target, triggering a recognition event. The requisite binding of two-components allows the probes to have excellent selectivity and modularity. The binary approach was applied to design a new sensor, called operating cooperatively (OC) sensor, for recognition of nucleic acids, including selectively differentiating between single nucleotide polymorphisms (SNPs). An OC sensor contains two oligonucleotide probe strands, called O and C, each with two domains. The first domain contains a target recognition sequence, whereas the second domain is complementary to a molecular beacon (MB) probe. Binding of both probe strands to the fully matched analyte generates a full MB probe recognition site, allowing a MB to bind and report the presence of the target analyte. Importantly, we show that the OC sensor selectively discriminates between single nucleotide polymorphisms (SNPs) in DNA and RNA targets at room temperature, including those with stable secondary structures. Furthermore, the combinatorial use of OC sensors to create a DNA logic gate capable of analyzing DNA sequences of Mycobacterium tuberculosis is described. The binary approach was also applied to design covalent inhibitors for HIV-1 reverse transcriptase (RT). In this application, two separate pre-reactive groups were attached to a natural RT ligand, deoxythymidine triphosphate (dTTP). Upon binding of both dTTP analogs in the RT active site, the pre-reactive groups are brought into the proper proximity and react with each other forming an intermediate that subsequently reacts with an amino acid side chain from the RT. This leads to covalent modification of RT, and inhibition of its DNA polymerase activity. This concept was tested in vitro using dTTP analogs containing pre-reactive groups derived from ?-lactamase inhibitors clavulanic acid (CA) and sulbactam (SB). Importantly, our in vitro assays show that CA based inhibitors are more potent than zidovudine (AZT), a representative of the dominant class of RT inhibitors currently used in anti-HIV therapy. Furthermore, molecular dynamics simulations predict that complexes of RT with these analogs are stable, and point to possible reaction mechanisms. The inhibitors described in this work may serve as the basis for the development of the first covalent inhibitors for RT. Moreover, the pre-reactive groups used in this study can be used to design covalent inhibitors for other targets by attaching them to different ligands. Overall, the work presented herein establishes the binary approach as a straightforward way to develop new probes to selectively recognize nucleic acids and proteins.