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published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Nucleic acids, as one of the most important macromolecules in living systems, play critical roles in storing, transferring, regulating genetic information, directing proteins synthesis, and catalysis. Understanding the structure of nucleic acid can bring us valuable information for mechanistic study and for drug discovery as well. Among all experimental methods, X-ray crystallography is the most powerful tool in structural biology study to reveal the 3D structure of macromolecules, which has provided over 80% of the highly detailed structural information to date. However, this great technology comes with two disturbing features, crystallization and phasing. The covalent selenium modification of nucleic acids has been proven to be a powerful tool to address both issues in nucleic acid crystallography. First part of this dissertation focuses on the development of novel selenium-modified nucleic acids (SeNA) for crystallization and phasing of B-form DNA containing structures. The novel 2’-SeMeANA modification is the first and currently the only selenium modification, which is fully compatible with X-ray crystallographic study of B-form DNA. Since selenium derivatization at 2’-arabino position dose not affect the B-type 2’-endo sugar conformation, this strategy is suitable for incorporating selenium into DNA for structural studies of B-DNA, DNA-protein complexes, and DNA-drug complexes. Specific base pairing is essential to many biological processes, including replication, transcription, and translation. It is crucial to NA (nucleic acid) sequence-based diagnostic and therapeutic applications as well. By utilizing the unique steric and electronic property of selenium, we designed, synthesized the novel 2-Se-U RNA modification, and demonstrated its highly specific base-pairing property by both biophysical and crystallographic methods. Our studies of 2-Se-U-containing RNAs suggest that this single-atom replacement can largely improve base pairing fidelity against U/G wobble pair, without significant impact on U/A pair.

Thesis advisor: Larry W. McLaughlin / Nucleic acids are fundamental biomolecules responsible for all activities of a living cell. DNA serves as an instruction manual to the cell, containing blueprints and directions for all cellular processes, while RNA serves to carry out the messages held within DNA. Research into the structure, stability, and function of nucleic acids has revealed much about the origin and evolution of life. The ultimate goal of this work is to understand how molecules bind and associate within the minor groove of double stranded, helical DNA. A series of 2'-deoxyadenosine analogues are modified at the three position by replacing the N3-nitrogen with carbon. Substitution at this position is designed to emulate the effects of removing hydrogen bond acceptors, introducing steric bulk, and tethering functional groups of interest into the minor groove. These functional groups mimic small molecules that have been shown to bind within the minor groove of A-T rich sequences as well as serve as a platform for further substitution by fluorescent tags. The synthetic effort needed to obtain purine nucleosides containing each of these modifications was non-trivial. New methodologies unveiled directing and protecting strategies towards the desired isomer of these modified nucleosides in higher yields than those previously deemed acceptable. Application of these modified nucleosides into duplex DNA reveals thermodynamic parameters for how small molecules bind to the minor groove and the effects of introducing biomarkers into an unprecedented region of DNA. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Zheng Ping-pin. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 118-159). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT(ENGLISH/CHINESE) --- p.iii / CONTENTS --- p.viii / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / PUBLICATION --- p.xiii / Chapter CHAPTER I --- INTRODUCTION / Chapter I.1. --- Preface --- p.1 / Chapter I.2. --- Overview of Carcinogenesis --- p.2 / Chapter I.3. --- Oncogene --- p.5 / Chapter I.4. --- Tumor Suppressor Genes (TSGs) --- p.6 / Chapter I.5 --- Detection of Oncogene and Tumor Suppressor Genes --- p.9 / Chapter I.5.1 --- Detaction of Oncogene --- p.9 / Chapter I.5.2. --- Detection of Tumor Suppressor Genes --- p.11 / Chapter I.6. --- Profiles of Oncogenes/TSGs and Molecular Subtype about Astrocytic Tumors --- p.17 / Chapter I.7. --- Intratumoral Heterogeneity and Microsatellite Instability --- p.20 / Chapter I.8. --- Outline of Ependymoma --- p.20 / Chapter I.9. --- Clinicopathological Factors and Prognosis --- p.22 / Chapter I.9.1. --- Histology and Grading (2000) --- p.22 / Chapter I.9.2. --- Prognosis Factors --- p.23 / Chapter I.9.2.1. --- Age/Sex/Location --- p.23 / Chapter I.9.2.2. --- Extent of Resection --- p.25 / Chapter I.9.2.3. --- Radiotherapy and Chemotherapy --- p.25 / Chapter I.9.2.4. --- Histology --- p.26 / Chapter I.10. --- "Cytogenetic, Molecular Genetic and Molecular Studies" --- p.27 / Chapter I.11. --- Advantages and Disadvantages of The Research Methods --- p.34 / Chapter CHAPTER II --- AIM OF STUDY --- p.36 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.37 / Chapter III.1. --- Tumor Samples and DNA Preparations --- p.37 / Chapter III.1.1. --- Tumor Samples --- p.38 / Chapter III.1.2. --- DNA Preparation --- p.38 / Chapter III.2. --- Comparative Genomic Hybridization --- p.42 / Chapter III.2.1. --- Metaphase Preparation --- p.42 / Chapter III.2.2. --- "DNA Labeling, Hybridization, and Detection" --- p.43 / Chapter III.2.3. --- Digital Image Analysis --- p.45 / Chapter III.3 --- High-Resolution Allelotying (Microsatellite Analysis) --- p.46 / Chapter III.3.1 --- General Outline --- p.46 / Chapter III.3.2 --- Multiplex PCR --- p.47 / Chapter III.3.3 --- Pooling of PCR Products --- p.49 / Chapter III.3.4 --- Electrophoresis --- p.50 / Chapter III.3.5. --- Assessment of Allelic Imbalance by Calculating Allelic Ratio --- p.52 / Chapter III.3.6 --- Standards of Evalution --- p.53 / Chapter III.3.7 --- Separating Allelic Loss from Allelic Duplication --- p.54 / Chapter III.3.8 --- Statistical Analyses --- p.54 / Chapter CHAPTER IV --- RESULTS --- p.54 / Chapter IV.1. --- CGH Study --- p.54 / Chapter IV.1.1 --- Overview --- p.54 / Chapter IV.1.2 --- Common Deletion Regions --- p.58 / Chapter IV.1.3 --- Common duplication Regions --- p.60 / Chapter IV.2. --- High-Resolution Allelotyping (Microsatellite Analysis) --- p.60 / Chapter IV.2.1. --- Overview of Results --- p.60 / Chapter IV.2.2. --- LOH profile of Individual Chromosome --- p.93 / Chapter IV.2.3. --- Overlapping Small Deletion Regions --- p.95 / Chapter CHAPTER V --- DISCUSSION --- p.97 / Chapter V.1. --- . General Outline --- p.98 / Chapter V.2. --- Chromosome 22 --- p.99 / Chapter V.3. --- Chromosome 17 --- p.102 / Chapter V.4. --- Chromosome 6 --- p.104 / Chapter V.5. --- Chromosome 16 --- p.105 / Chapter V.6. --- Chromosome 19 --- p.107 / Chapter V.7. --- Chromosome 20 --- p.108 / Chapter V.8. --- Chromosome 7 --- p.109 / Chapter V.9. --- Chromosome 12 --- p.110 / Chapter V.10. --- Chromosome 9 --- p.111 / Chapter V.11. --- Chromosome 5 --- p.112 / Chapter V.12. --- Chromosome 4 --- p.112 / Chapter V.13. --- Correlation of CGH with Allelotyping in the Study --- p.112 / Chapter V.14. --- Conclusion --- p.114 / Chapter CHAPTER VI --- LIMITATIONS OF THE STUDY --- p.115 / Chapter CHAPTER VII --- FUTURE STUDY --- p.116 / REFERENCES --- p.118

Ribonucleases play essential roles in RNA metabolism. In Escherichia coli, the extensive degradation of RNAs that are defective or no longer required by the cell is carried out by one of three processive, 3' to 5' exoribonucleases. Relatively unstructured mRNAs are typically degraded by RNase II or PNPase. In contrast, mRNAs containing extensive secondary structure, and the highly structured rRNA and tRNA molecules, are degraded by PNPase and/or RNase R. However, RNase R differs from other exoribonucleases in that it is able to degrade through these structured RNAs without the aid of a helicase activity. Consequently, its mechanism of action is of great interest. In this dissertation, using a variety of specifically designed substrates, I show that a single-stranded overhang, which must be at least 5 nucleotides in length, is required for tight binding and subsequent degradation of double-stranded RNA by RNase R. Moreover, this overhang must be 3' to the duplex indicating that an RNA substrate must thread into the enzyme with 3' to 5' polarity. Using a series of truncated RNase R proteins, I show that the cold-shock domains and the S1 domain contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, while the S1 domain is required to position substrates for efficient catalysis. Furthermore, the nuclease domain alone is sufficient to bind and degrade structured RNAs. This is a unique property of the nuclease domain of RNase R since this domain in RNase II, a paralogue of RNase R, stalls as it approaches a duplex. RNase R binds RNA more tightly within its nuclease domain than RNase II. Through mutagenesis studies, I identify one amino acid, R572, within the nuclease domain of RNase R that contributes to this tight binding and the ability to degrade double-stranded RNA. Furthermore, I found that degradation of structured RNA is strongly dependent on temperature. Based on these data I propose that tight binding allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNA.

Detection of cellular messenger RNA is a useful diagnostic strategy for the detection of patho-gens. A rapid and sensitive method for on-site detection of specific pathogens would be of great use in a number of fields. For example, a simple and inexpensive method for the detection of harmful biological agents in train stations and airports is useful for national security. Rapid detection of pathogenic E. coli strains in food production would also be of great benefit in ensuring the safety and quality of our food supply. Here we present a method for the rapid de-tection of cellular mRNA. This system is based on the 3’-labeling approach in which targeted RNA is simultaneously extended and labeled with the use of biotin labeled-dNTPs and DNA po-lymerase on an immobilized nucleic acid probe. The biotin is subsequently converted to an enzymatic label, which produces a detectable chemiluminescent reaction in the presence of substrate. Detection time of this system is short (approximately 20 minutes) because there is no need for amplification by PCR, transcription, or fluorophore labeling. This novel methodology has been successfully demonstrated by selective detection of lac Z mRNA in a total RNA sample from E. coli.

The detection of particular genetic sequences aids in the early detection and diagnosis of disease; permits monitoring of the health and state of the natural environment; and informs forensic investigations. To date, gene detection has relied on enzymatic amplification followed by optical readout. Though these technologies have advanced dramatically, the instruments and assays are costly and lack portability. The work presented herein addresses an urgent challenge: molecular diagnostics at the point-of-need. This work reports the first electronic chip capable of analyzing - directly, without amplification, and with clinically-relevant sensitivity - multiple genes of interest present in a clinical sample. It reports a dramatic acceleration in sample-to-answer times, with clinically actionable findings in minutes where legacy techniques take hours or days. The key to the sensitivity and speed of the biosensors reported herein lies in their architecture and morphology on multiple lengthscales. It is proven that hybridization-based assays employing a nucleic probe attached to a solid surface can only achieve efficient performance when displayed on a nanotextured surface. It is also discovered that these same sensing elements must reach tens of micrometers into solution to achieve rapid, sensitive detection of nucleic acids in clinical samples. As a result, the materials integrated onto the sensing chip reported herein are engineered on multiple lengthscales - from the nanometers to the tens of micrometers. Engineering is done through a combination of low-cost, convenient top-down photolithographic patterning; combined with hierarchically-designed bottom-up growth of electrodeposited sensing elements. The capstone of this work is a chip that distinguishes among different types of bacteria in an unpurified sample. The chip gives accurate answers in under half an hour when detecting bacteria at a level of 1.5 colony-forming-unit (cfu) per microliter. These speeds and sensitivies enable the application of this technology in point-of-need assays for infectious disease detection. Ultimately, the work showcases the power of bringing together techniques and principles from materials chemistry, biochemistry, applied physics, and electrical engineering to the solution of an important problem relevant to human health.

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.

The detection of particular genetic sequences aids in the early detection and diagnosis of disease; permits monitoring of the health and state of the natural environment; and informs forensic investigations. To date, gene detection has relied on enzymatic amplification followed by optical readout. Though these technologies have advanced dramatically, the instruments and assays are costly and lack portability. The work presented herein addresses an urgent challenge: molecular diagnostics at the point-of-need. This work reports the first electronic chip capable of analyzing - directly, without amplification, and with clinically-relevant sensitivity - multiple genes of interest present in a clinical sample. It reports a dramatic acceleration in sample-to-answer times, with clinically actionable findings in minutes where legacy techniques take hours or days. The key to the sensitivity and speed of the biosensors reported herein lies in their architecture and morphology on multiple lengthscales. It is proven that hybridization-based assays employing a nucleic probe attached to a solid surface can only achieve efficient performance when displayed on a nanotextured surface. It is also discovered that these same sensing elements must reach tens of micrometers into solution to achieve rapid, sensitive detection of nucleic acids in clinical samples. As a result, the materials integrated onto the sensing chip reported herein are engineered on multiple lengthscales - from the nanometers to the tens of micrometers. Engineering is done through a combination of low-cost, convenient top-down photolithographic patterning; combined with hierarchically-designed bottom-up growth of electrodeposited sensing elements. The capstone of this work is a chip that distinguishes among different types of bacteria in an unpurified sample. The chip gives accurate answers in under half an hour when detecting bacteria at a level of 1.5 colony-forming-unit (cfu) per microliter. These speeds and sensitivies enable the application of this technology in point-of-need assays for infectious disease detection. Ultimately, the work showcases the power of bringing together techniques and principles from materials chemistry, biochemistry, applied physics, and electrical engineering to the solution of an important problem relevant to human health.