I am interested in the design of DNA binding ligands which are including DNA minor groove binders and G-quadruplex stabilizing agents. Certain natural products, distamycin and belomycin are considered as models for designing new DNA binding agents. A variety of DNA binding ligands were synthesized and accordingly characterized by different bioassays. In the series of azo-pyrrole-polyamide, it showed slight DNA binding affinity but has the properties of DNA photo-cleavage and recognition of mixed sequence. The thiophene series showed strong binding ability for duplex DNA. Bithiophene series showed a remarkable binding affinity to duplex DNA which is compatible to natural products netropsin and distamycin. The pyridodicarboxamide series remain intramolecular hydrogen bonding with poor preferences for duplexes DNA even though they stabilized quadruplex DNA. The bithiophene-bipyrimidine compound binds to specific hexanucleotide sequence 5¡¦-AAGCTT-3¡¦ and acts as an unfused quadruplex stabilization ligand. In the quinoline series, the combination of quinoline and methylpyrrole polyamide proves the attenuation effect for quadruplexes stabilization which provides a novel strategy for development of quadruplex binding logands. In this study, several series of small molecules for DNA binding have been successfully synthesized and proved to interact with DNA secondary structures according to the particular properties of themselves.

Certain natural products, Ditamycin and Netropsin are considered as models of designing new DNA binding agents. A variety of DNA binding ligands were synthesized and accordingly characterized by different bioassays. In the series of azo-polyamide, it showed slight DNA binding affinity but has the properties of DNA photo-cleavage and recognition of mixed sequence. The carbohydrate-azo-polyamide series show the properties of DNA photo-cleavage and more effective in vitro experiment. In the quinoline series proves the attenuation effect for G-quadruplex stabilization which provides a novel strategy for development of G-quadruplex binding ligands.

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

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

In vitro, G-rich sequences form highly stable secondary structures known as G-Quadruplexes. These structures have been characterized by circular dichroism nuclear magnetic resonance and X-ray crystallography; although their detection in vivo has remained elusive. Due to the biological implication of a transisent and polymorphic secondary structure forming within the hypothetical G-Quadruplex forming regions, there is growing interest to understand their in vivo molecular dynamics. / text

Two diverse works regarding DNA-Drug Interaction are presented here. The first portion deals with covalent interactions between compounds that are derivatives of heterocyclic aza-enediynes and DNA (conventional Watson-Crick base paired double stranded DNA) and the second is related to non-covalent interactions of these compounds with G-quadruplex DNA. The aza-enediynes have been studied for their ability to undergo aza-variants of the Bergman and Myers cyclizations, and the potential role of the ensuing diradicals in DNA cleavage chemistry. The aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes has been recently reported. In the first part of the dissertation, the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-Npropargyl pyridine salts) are reported. Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. An optimized analog containing a p-methoxyphenyl substituent was prepared. Studies with radiolabeled DNA duplexes reveal that this analog generates nonselective frank DNA strand breaks, via deoxyribosyl 4'-hydrogen atom abstraction, and also leads to oxidation of DNA guanine bases. This is the first report of enediynelike radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization. The second part is based upon the growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for replication. A prototypical replicative helicase - SV40 large T-antigen (T-ag), a multifunctional protein with duplex DNA helicase activity is shown to also unwind G-quadruplex DNA structures. A series of G-quadruplex-interactive agents, particularly perylene diimide derivatives, is explored for inhibition of T-ag duplex and G-quadruplex DNA unwinding activities, and it is revealed that certain perylene diimides are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic Gquadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.

In this dissertation, the formation of unusual G-quadruplexes in the critical regions of the c-myb and hTERT promoters for control of promoter activity was investigated.The c-myb promoter contains three copies of an almost perfect (GGA)4 sequence. We demonstrate that the each (GGA)4 repeat forms a tetrad:heptad G-quadruplex and any two of the three can intramolecularly dimerize to form T:H:H:T G-quadruplexes. The three T:H:H:T G-quadruplex combinations are of differing degrees of stability and can be further stabilized by G-quadruplex interactive compounds. We also demonstrate that the c-myb G-quadruplex forming region is a critical transcriptional regulatory element and interacts with various nuclear proteins including MAZ (Myc Associated Zinc finger protein). The data from luciferase reporter assay show that the c-myb GGA repeat region plays dual roles as a transcriptional activator and an inhibitor by serving as binding sites for the activators and by forming G-quadruplex structures in the region, respectively. Furthermore, we show that MAZ is a transcriptional repressor of the c-myb promoter and binds to both the double-stranded and T:H:H:T G-quadruplex-folded conformations of the GGA repeat region of the c-myb promoter.The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, which includes three critical Sp1 binding sites. Although this G-rich region has the potential to form multiple G-quadruplexes, our investigation on the full-length G-rich sequence demonstrate that the G-rich region forms a unique G-quadruplex structure in which two tandem intramolecular G-quadruplex structures are present, consisted of one G-quadruplex formed by the G-tracts 1-4 and the other formed by the G-tracts 5, 6, 11, and 12. We also demonstrate that the latter unusual structure contains a 26-base middle loop that likely forms a hairpin structure and is more stable than the other conventional G-quadruplex. Significantly, the formation of this unusual tandem G-quadruplex structure in the full-length will disable all three critical Sp1 binding sites, which will dramatically downregulate hTERT expression. G-quadruplex formation in the hTERT promoter suggests that the effect of G-quadruplex interactive ligands on telomerase inhibition and telomere shortening may be exerted by the direct interaction between the hTERT G-quadruplex structure and the ligands.

Although some progress has been made in the treatment of cancer over the last sixty years, the majority of chemotherapeutics has fallen short. Because general chemotherapies that target DNA replication have only a limited efficacy and significant non-target side-effects, a new paradigm for cancer drug development has been adopted. Using a molecular targeted approach, new gene and protein targets have been identified and the development of chemotherapies that are specific to these targets has already begun. In this study, compounds that interact with two key cancer targets, the G-quadruplex of the c-Myc promoter and p-glycoprotein, have been investigated. By developing such compounds, improvements in treatment efficacy is anticipated with an aspiration for decreased mortality attributable to cancer.Formation of DNA secondary structures, such as the G-quadruplex, in the NHE III1 region of the c-Myc promoter has been shown to repress c-Myc transcription. Because c-Myc is an oncogene that is overexpressed in a variety of cancers, stabilization of the G-quadruplex by small molecules would be advantageous in cancer treatment. Using Fluorescence Resonance Energy Transfer, with Taq Polymerase Stop assays for confirmation, a group of compounds were identified that stabilize the c-Myc G-quadruplex structure. Using a colon cancer model, two compounds were shown to decrease c-Myc gene and protein expression. Also, exposure to the compounds for 48 hours results in an induction of caspase-3, indicative of apoptosis. Furthermore, surface plasmon resonance suggests that compound-induced stabilization of the c-Myc G-quadruplex can prevent sustained binding of the regulatory protein NM23-H2 by increasing its dissociation from the G-quadruplex. This may subsequently prevent unraveling of the G-quadruplex.Because resistance to chemotherapy reduces its effectiveness, development of multidrug resistance (MDR) modulators was also studied. Psorospermin is a topoisomerase II-directed DNA alkylating agent active against MDR cell lines. In a study examining the mechanism of psorospermin's P-glycoprotein modulation, Flow Cytometry demonstrated that doxorubicin-resistant multiple myeloma cells pre-treated with psorospermin enhanced intracellular retention of doxorubicin. Because neither transcription of mdr1 nor translation of P-glycoprotein was downregulated by psorospermin, resistance reversal is most likely due to a direct interaction between the side chain of psorospermin and P-glycoprotein, inhibiting drug efflux.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

An increasing interest in non-canonical nucleic acid structures has drawn the attention of the scientific community during the last few decades. One such structure, the G-quadruplex, has been the focus of an increasing number of scientists as G-quadruplexes are believed to play a role in biological processes such as telomere integrity and gene expression. Their existence in vivo is largely unproven but they have stimulated a lot of research into small molecules that interact with them. The development of a new class of such molecules is described in this thesis. A member of this family showed to be very selective in stabilising one particular G-quadruplex. The further development of another family of G-quadruplex interacting small molecules is also presented in this thesis and some of their effects in cellulo were assessed. Based on the scaffold of this family, an affinity probe was developed, which can mediate the isolation of its nucleic acid targets from human cells. This is the first example of the use of a small molecule with an affinity tag that has been used to isolate a nucleic acid target in a structure specific manner from human cells.