Doctor of Philosophy

dissertationLittle is known about the kinetic limitations of the polymerase chain reaction (PCR). Advancements in chemistry and instrumentation have increased its speed and specificity. Further improvements will be facilitated by a more complete understanding of the rates of the individual reactions that comprise PCR. A continuous fluorescent assay is developed to study DNA polymerase extension. Nucleotide incorporation is monitored with noncovalent DNA dyes using a defined hairpin template. The extension rate is measured in nucleotides incorporated per second per molecule of polymerase and has greater relevance to PCR than traditional activity methods. This assay was developed and validated on a stopped-ow instrument and subsequently adapted for real-time PCR instruments to extend its utility to any laboratory setting. The influences of a variety of buer components were determined and optimal conditions for fast polymerase extension are recommended. The incorporation rates of each nucleotide were determined and extension was found to depend on template sequence. When DMSO was included in the reaction to reduce inhibition from secondary structure, extension rates of random sequences were closely approximated by their base composition. Extension rates as a function of temperature were determined and were applied to a kinetic model. This model accounts for extension during temperature transitions and more accurately portrays fast PCR with rapid thermal cycling. A complete model of PCR based on differential equations derived from mass action equations is provided. This can be used to incorporate experimentally derived parameters obtained for the other reactions of PCR. Knowledge of the temperature and chemistry dependence of reaction rates will enable improved thermal cycling and solution conditions for more rapid and effcient PCR

Engineering the central dogma using emulsion based directed evolution

The central dogma of molecular biology forms the most basic (and fundamental) paradigm of how life operates. Despite its elegant simplicity, scientists are still uncovering enigmas of the central dogma - which has been shaped throughout billions of years of the Darwinian process. Even though the core concepts of the central dogma have largely been untouched by evolution (the universality of the genetic code, amino acid utilization, DNA/RNA base identity) scientific advances have shown that these fundamental properties can be altered dramatically. This implies the architectures of life are pliable and likely the result of extreme optimization and fine tuning of semi-random events that took place soon after the origin of life. Reengineering the parameters of life offers a unique way of testing evolutionary processes and perceived optimality of its components. Naturally, coaxing proteins and nucleic acids to function in an unnatural fashion is difficult. Development of techniques to enable these changes has relied heavily on the exploitation of water-in-oil emulsions (or, in vitro compartmentalization), which allows directed evolution at the single cell or even single molecule level. In particular, compartmentalized partnered replication (CPR) is a dual mode selection technique, coupling the in vivo functionality of a gene with the in vitro amplification via emulsion PCR. The CPR technique has enabled the development of synthetic promoter recognition by T7 RNA polymerase, unnatural amino acid incorporation by aminoacyl tRNA synthetase engineering, genetic code reassignment through tRNA evolution, and transcriptional regulation using repressors with novel allosteric effector molecules and operator binding sites. Using a similar technique, the template recognition of an Archaeal DNA polymerase was altered such that the polymerase utilizes both DNA and RNA templates with similar efficiencies. This resulted in a reverse transcriptase that can functionally proofread on RNA templates. These technologies will continue to play a pivotal role in the future development of particular aspects of the central dogma. As certain steps in this process are tweaked to have alternative functionalities and combined together, the gap between natural life and synthetically modified life widens and gives the Darwinian process of evolution new areas to explore.Cellular and Molecular Biolog

Synthesis, characterizations and applications of C2'-modified oligonucleotide analogues

During the past two decades, oligonucleotide analogues have drawn considerable attention as potential therapeutic and diagnostic agents. Gene silencing through "RNA interference" (siRNA) or the more mature "antisense" technology (AONs) have proven to be powerful tools for studying gene functions. Chemical modifications of these compounds are generally required to improve their "drug-like" properties such as potency, selectivity and delivery, particularly in the development of oligonucleotide-based therapeutics. Aptamers are another emerging class of oligonucleotide therapeutics and diagnostics.This thesis focuses on oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta- D-ribofuranosyl)thymine (2'-alpha-hm-dT, abbreviated as "H") and 2'-deoxy-2'-fluoroarabinonucleotides (2'F-araN), and their applications. A major component of this work focused on the synthesis of 2'-alpha-hm-dT (H) and the first investigation of oligoribonucleotides containing this nucleoside analogue. Specifically, 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. Incorporation of 3',5'-linked H units into a DNA, 2',5'-RNA or RNA strand led to significant destabilization of duplexes formed with unmodified RNA targets. 2',5'-Linked H units into 2',5'-RNA or RNA caused significantly less destabilization, and in fact, they were shown to stabilize the loop structure of some RNA hairpins. These results were rationalized in terms of the "compact" and "extended" conformations of nucleotides.A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and nonnatural (i.e., H, and acyclic nucleoside) branch points in order to examine the effect of sugar conformation and phosphodiester configuration on yeast debranching enzyme (yDBR) hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a ribose-phosphate backbone at the branch point, whereas some of the H-containing branched RNAs were found to competitively inhibit yDBR hydrolytic activity.This thesis also examines the stabilization of DNA guanine-quadruplexes (G-quadruplexes) by replacing the deoxyribose sugar by a 2-deoxy-2-fluoroarabinose. The effect of this substitution was assessed in the well-known thrombin-binding DNA aptamer d(G2T2G2TGTG2T 2G2), the telomeric DNA d(G4T4G 4) sequence and a phosphorothioate octanucleotide PS-d(T2G 4T2), all of which are known to fold into G-quadruplex structures. Stabilization of the G-quadruplexes was possible provided that the arabinose sugar was introduced at guanosine residues adopting an anti N-glycosidic bond conformation. Some of the arabinose modified thrombin-binding aptamers not only exhibited superior thermal stability and nuclease resistance, but also maintained high thrombin binding affinity.Finally, this thesis examines the ability of DNA polymerases to recognize and utilize 2'-deoxy-2'-fluoro-beta-D-arabinonucleoside 5'-triphosphates (2'F-araNTPs) as building blocks for the synthesis of 2'-deoxy-2'-fluoro-beta- D-arabinonucleic acids (2'F-ANA). The results obtained indicate that a few DNA polymerases can synthesize 2'F-ANA and 2'F-ANA-DNA chimeras on a DNA template. Conversely, certain enzymes were shown to catalyze 2'F-ANA template-directed DNA synthesis. While it was not possible to synthesize 2'F-ANA strands on a 2'F-ANA template, it is possible for some DNA polymerases to catalyze the formation of multiple 2'F-ANA:2'F-ANA base pairs within a DNA-FANA chimeric duplex. These results suggest that it should be possible to evolve FANA-modified aptamers via SELEX

Chemical synthesis and enzymatic incorporation of artificial nucleotides

Deutsche Übersetzung des Titels: Chemische Synthese und enzymatischer Einbau von künstlichen Nukleotide

BIOCHEMICAL STUDY OF ENDONUCLEASE V AND ITS APPLICATION IN MUTATION SCANNING

The integrity of the genetic information encoded by DNA is essential to all living organisms, yet the reactive bases of DNA are constantly attacked by endogenous and exogenous agents resulting in as many as one million individual molecular lesions per cell per day. Excessive DNA damage or deficiency in DNA repair enzymes may cause cancer, premature aging, and neurodegenerative diseases. Endonuclease V (Endo V) is a DNA repair enzyme which can recognize all four types of DNA deamination products, specifically, uracil, hypoxanthine, xanthine and oxanine. It was also shown that endo V can recognize mismatches. We screened about 60 mutants of endo V from Thermotoga maritima and found some mutants had altered base preferences for mismatches. Tma endo V Y80A was shown to become a C-specific mismatch endonuclease. G13D mutation in K-ras oncogene which was not recognized by wild type Tma endo V was successfully cleaved by Tma endo V Y80A. This study provides valuable information on base recognition and active site organization of Tma endo V. Tma endo V mutants can be used for cancer mutation scanning and mutation recognition. In order to further understand the role of Y80 of endo V in base recognition, we substituted the Y80 with sixteen amino acids. Together with three Y80 mutants isolated before, we characterized all nineteen mutants of Tma endo V Y80 using deaminated base-containing DNA substrates and mismatch-containing DNA substrates. This comprehensive amino acid substitution at a single site (Y80) underlines the importance of aromatic ring and hydrogen bond donor capacity in base recognition by endo V, reveals additional Y80 mutants with altered base preferences in mismatch cleavage, and offers new insight on the role of Y80 in base recognition. Though endo V was shown to be important for repair of deaminated lesions in vivo, its DNA repair pathway remains unknown. In order to understand the DNA repair pathway mediated by endo V, we have developed a cell-free system from Escherichia coli. The preliminary results indicated that the repair patch of endo V mediated DNA repair pathways may consist of a long patch and a short patch repair pathway

The ligase detection reaction: the evolution of a mutation detection strategy

Early detection of genetic mutations is important for control of diseases such as cancer and Alzheimer\u27s. Early detection requires methods that detect small amounts of mutated DNA in very large amounts of normal or wild type DNA. One method to detect mutated DNA is the ligase detection reaction (LDR). Since its inception LDR has evolved greatly from a simple detection reaction after PCR amplification to PCR/RE/LDR, a scheme which uses nucleoside base analogs in PCR to convert wild type sequences to sequences containing restriction endonuclease (RE) sites which can then be cleaved leaving only mutant sequences for detection by LDR. Analysis of LDR has also evolved from slab gel electrophoresis to microarray analysis. Understanding the structure and DNA polymerase recognition of nucleoside base analogs used in PCR/RE/LDR is key to improving this detection scheme. The use of higher fidelity DNA polymerase containing 3\u27→5\u27 exonuclease domains for error correction is also important in early detection of genetic diseases. Pyrazole-based nucleoside analogs have been studied computationally and enzymatically. The stability a DNA containing these analogs depends largely on the dipole moment of the analogs, rather than polarizability or surface area. Reduced DNA polymerase recognition is due in part to altered base pair geometry, either inherent or created by DNA polymerase. Thiazole and thiazole N-oxide analogs to be used in the PCR/RE/LDR assay have been synthesized and characterized computationally, thermodynamically, and enzymatically. The N-oxide, a pyrimidine O2 mimic, enhances DNA stability and DNA polymerase recognition. The N-oxide increases electrostatic properties and solvation by the formation of a hydrogen bond when base paired with guanine. Enzymatic analysis indicated a preference for the base pairing of thiazole N-oxide with guanine and thiazole with adenine. An N3\u27→P5\u27 phosphoramidate backbone analog has shown to inhibit the exonuclease activity of higher fidelity DNA polymerases for use in PCR/RE/LDR. The evolution of the analysis of LDR continues with the adaptation to capillary and microdevice electrophoresis. These formats were used to analyze model samples and LDR reactions mimicking low abundant mutations. These improved techniques greatly improve the resolution of LDR analysis

Characterization of the multifunctional XPG protein during Nucleotide-excision-repair

Xeroderma pigmentosum (XP), a cancer model disease, is the perfect proof for the existing model of carcinogenesis activated by mutations. All patients share a defect in Nucleotide excision repair (NER). The gene, which is disease-causing for XP complementation group G (XPG) patients, encodes for the multifunctional endonuclease XPG. This enzyme has many binding partners like TFIIH, RPA and PCNA, and acts at a crucial step at the very end of NER. Several functional domains of XPG were mutated to investigate the behavior of the respective mutants during NER intermediates of dual incision, using DNA repair synthesis (UDS) and Host cell reactivation (HCR) assays. Furthermore, a new XPG patient with implications for the functional XPG-TFIIH interaction has been studied. By genotype-phenotype correlation of a XPG patient (XP172MA), this study greatly suggests to narrow down the functionally important XPG interaction domain between TFIIH and XPG to the XPG amino-acids 30-85. This study demonstrates that the functional PCNA-XPG interaction is more important for NER than the endonuclease function of XPG. The C-terminally located PIP-box of XPG is required for immediate UV response but not for the functionality of XPG during NER in transiently transfected primary fibroblasts. The N-terminal PIP-UBM ubiquitin binding domain is more important for integrity of NER than the C-terminal PIP-box. I raise the model of an NER intermediate state that involves obligatory ubiquitination during NER and the blocking of error-prone translesion polymerases by XPG. This study excludes XPG as the responsible factor for PCNA recruitment and designates XPG as the factor as restrictive element for UV-damage dependent activation of translesion polymerases to S-phase. The results obtained with the endonuclease defective E791A XPG mutant confirm the actual “cut-patch-cat-patch” model of dual incision during NER. Moreover, this study clearly demonstrates the ability of endonuclease defective XPG to perform accurate NER in living cells. This accounts for the existence of a cellular backup mechanism for the XPG endonuclease function. The proposal for a nuclear backup mechanism is supported by the investigation of a physiologically relevant (evolutionary developed) XPG splicevariant with NER activity (IsoVI). The severely truncated XPG isoform is able to structurally complement a XPG defect. This complementation is dependent on the endonuclease function of Fen1. This suggests the existence of an evolutionary developed backup mechanism for XPG during NER

Chemical synthesis and enzymatic incorporation of artificial nucleotides

Deutsche Übersetzung des Titels: Chemische Synthese und enzymatischer Einbau von künstlichen Nukleotide

An investigation into the role, recruitment, and regulation of PrimPol in DNA replication restart

DNA replication is one of life's fundamental processes. This delicate task is performed by a complex of molecular machines, known collectively as the replisome. At the heart of the replisome lie the replicative DNA polymerases which catalyse synthesis of daughter DNA strands with astonishing accuracy and efficiency. Nevertheless, these enzymes are prone to stalling upon encountering DNA damage lesions and secondary structures. In order to restart replication, DNA damage tolerance mechanisms are required. This published article-format thesis focusses on a recently discovered primase-polymerase, and member of the archaeo-eukaryotic primase (AEP) superfamily, involved in DNA damage tolerance, known as PrimPol. The work presented here builds on the initial characterisation of the enzyme, which identified potential roles in the bypass of DNA damage through translesion synthesis (TLS) and repriming of replication. The first presented article consists of a review of the AEP superfamily, functionally repositioning the group under the category of primase-polymerases. In the second paper, evidence is presented to suggest that PrimPol's activity is regulated by single-strand binding proteins, required due to the enzyme's error-prone nature. In the third chapter, a novel PrimPol binding protein, polymerase delta-interacting protein 2 (PolDIP2), is identified and characterised as a stimulatory factor for PrimPol's primer extension activities. Chapter 4 focusses on the development and use of a gel-based fluorescent primase assay to assess PrimPol's ability to reprime downstream of DNA damage lesions and secondary structures. The fifth presented paper identifies the molecular basis for PrimPol's interaction with replication protein A (RPA). Using biophysical, biochemical, and cellular approaches, this paper identifies the mechanism by which PrimPol is recruited to reprime replication. Lastly, in Chapter 6, a review of the presented articles in the context of the wider literature is included. Together, this work supports a role for PrimPol in repriming and restarting DNA replication following stalling at impediments, as well as identifying mechanisms involved in the recruitment and regulation of the enzyme

Studies on Boronic Acid-modified Nucleotides for Diagnostic Applications and Development of Fluorescent Chemoprobes for Molecules of Biological Importance

Post-synthesis DNA modification is a very useful method for DNA functionalization. This is achieved by using a modified NTP, which has a handle for further modifications, replacing the corresponding natural NTP in polymerase-catalyzed DNA synthesis. Subsequently, the handle can be used for further functionalization, preferably through a very fast reaction. Herein we describe polymerase-mediated incorporation of trans-cyclooctene modified thymidine triphosphate (TCO-TTP). Subsequently, the trans-cyclooctene group was reacted with a tetrazine tethered to other functional groups through a very fast click reaction. The utility of this DNA functionalization method was demonstrated with the incorporation of a boronic acid group and a fluorophore. The same approach was also successfully used in modifying a known aptamer for fluorescent labeling applications. Boronic acid modified DNA molecules were further applied in aptamer selection for cancer cell recognition. The second project was focused on developing fluorescent probes/sensors for biothiols. Because of the biological relevance of thiols and sulfides such as cysteine, homocysteine and hydrogen sulfide, their detection has attracted a great deal of research interest. Fluorescent probes are emerging as a new strategy for thiol and hydrogen sulfide analysis due to their high sensitivity, low cost, and ability to detect and image thiols in biological samples. A sulfonyl azide-based fluorescent probe has been developed for the quantitative detection of H2S in aqueous media such as phosphate buffer and bovine serum. In addition, another novel fluorescent probe has been developed for the detection of homocysteine. The fluorescence response is selective for homocysteine over other biologically abundant thiols such as cysteine and glutathione. In addition, a linear calibration curve was also be obtained for quantitative analysis in phosphate buffer and plasma. These selective fluorescent probes could be very useful tool for biothiol tests