Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

G-quadruplexes represent a versatile sensing platform for the construction of label-free molecular detection assays due to their diverse structures that can be selectively recognized by G-quadruplex-specific luminescent probes. In this thesis, we have explored the applications of the label-free G-quadruplex-based luminescent detection platforms for the detection of biomolecules and metal ions. Chapter 1 provides an overview of the principles and recent developments of the field of luminescent oligonucleotide-based probes, and highlighting in particular the use of the “label-free” strategy for the construction of simple and inexpensive sensing platforms. Chapter 2 introduces the basic experiments performed during the course of this thesis, including UV/Vis absorption spectroscopy, luminescence spectroscopy, nuclear magnetic resonance, mass spectrometry circular dichroism spectroscopy and G-quadruplex fluorescent intercalator displacement assay. Chapter 3 describes a G-quadruplex-based switch-on luminescence assay for the detection of gene deletion using iridium(III) complex 1 as a G-quadruplex-selective probe. Our method is based on the formation of a split G-quadruplex upon hybridization of two critically designed quadruplex-forming sequences with the mutant DNA sequence, resulting in a “switch-on” luminescence response. Chapter 4 describes a label-free, oligonucleotide-based, switch-on luminescence detection method for T4 polynucleotide kinase activity using a G-quadruplex-selective luminescent iridium(III) complex 2. The application of the assay for screening potential T4 PNK inhibitors is also demonstrated. To our knowledge, this is the first metal-based assay for PNK activity that has been reported in the literature. Chapter 5 describes a label-free oligonucleotide-based luminescence switch-on assay for the selective detection of sub-nanomolar Pb2+ ions in aqueous solution and real water samples. Iridium(III) complex 1 was employed as a G-quadruplex-specific luminescent probe and a guanine-rich DNA sequence (PS2.M, 5.-GTG3TAG3CG3T2G2-3.) was employed as recognition unit for Pb2+ ions. The assay could detect Pb2+ ions in aqueous media with a limit of detection of 600 pM, and also exhibited good selectivity for Pb2+ ions over other heavy metal ions. Furthermore, the application of the assay for the detection of Pb2+ ions in spiked river water samples was demonstrated. Chapter 6 describes a label-free G-quadruplex-based luminescent switch-on assay for the selective detection of micromolar histidine in aqueous solution. Iridium(III) complex 8 was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5.-TG4AG3TG4AG3TG4A2G2-3.)/cupric ion (Cu2+) ensemble was employed as a recognition unit for histidine. The assay could detect down to 1 µM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine-masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated. Chapter 7 summarizes the work that was conducted in this thesis, and the future outlook of G-quadruplex-based sensing is presented.

Sepsis is among the most prevalent diagnosed critical illnesses in the United States today. Although advances have reduced the overall morbidity and mortality associated with this illness, the enormous number of deaths associated with it shows a need for improved diagnostic and therapeutic optionsgent. Our laboratory has utilized RNA based technologies to aid in the treatment of histone induced multiple organ dysfunction syndrome seen in sepsis. Histones are proteins found in the nucleus of every cell in our body and have been shown to be released during sepsis. Such release induces damage to other cells, causing a feed forward cycle that results in organ failure and death. Several therapeutics have been utilized to neutralize histones but have shown considerable toxicity. This thesis describes the generation of single stranded RNA aptamers to bind and neutralize histone mediate damage without unwanted toxicity. We demonstrate that our aptamers selectively bind to histones but not serum proteins. In addition, we establish that our aptamers can neutralize all histone mediated cellular response in vitro and in vivo. Finally, we determined that our aptamers are able inhibit the histone feed forward cycle in a temporal fashion in our murine model of multiple organ dysfunction. This novel therapeutic demonstrates the selectivity and effectiveness needed to inhibit histones in several critical illnesses.

No description available.

Oligonucleotides may become a new class of therapies with the potential of curing many today untreatable diseases. Oligonucleotides becomes increasingly more difficult to separate with an increase in length since the relative difference in retention of these very similar compounds becomes increasingly smaller. Therefore, coelution of impurities formed during synthesis may result in insufficient purity, which is necessary for therapeutic treatments. Oligonucleotides are also relatively large biomolecules, possibly consisting of hundreds of nucleotides. As a result, oligonucleotides may have limited diffusion through the stationary phase pores which affects separation performance. Surprisingly few studies have be published in this research area and a wider knowledge in how this affects separation is needed. In this master thesis, separation of deoxythymidine oligonucleotides with 5-30 mers in length were separated with 60, 100, 200 and 300 Å pore size reversed phase C4 columns. It was concluded that pore size resulted in more restricted diffusion if insufficient pore size was used. Poor peak performance was also observed with too large pore sizes which lead to less efficient separations.

Lau Pui Man Irene. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 162-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Figures --- p.ix / List of Tables --- p.xii / Contents --- p.xiii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.1.1. --- Therapeutic uses of nucleic acids --- p.2 / Chapter 1.1.1.1 --- Antisense oligonucleotides --- p.2 / Chapter 1.1.1.2 --- RNA interference --- p.4 / Chapter 1.1.1.3 --- Aptamer --- p.6 / Chapter 1.2 --- Selection of Aptamer --- p.7 / Chapter 1.2.1 --- SELEX 'Systematic Evolution of Ligands by Exponential enrichment' --- p.7 / Chapter 1.2.1.1 --- In vitro selection --- p.8 / Chapter 1.2.1.2 --- Amplification --- p.8 / Chapter 1.2.1.3 --- Monoclonal Aptamer --- p.10 / Chapter 1.2.2 --- Photo-SELEX --- p.10 / Chapter 1.3 --- Examples of target molecules of aptamers --- p.12 / Chapter 1.4 --- Applications of aptamer --- p.14 / Chapter 1.4.1 --- Detection of Aptamer --- p.14 / Chapter 1.4.2 --- Examples of diagnostic use Contents --- p.15 / Chapter 1.4.2.1 --- Aptamer against theophylline with high specificity --- p.15 / Chapter 1.4.2.2 --- Aptamer chip --- p.16 / Chapter 1.4.3 --- Examples of therapeutic use --- p.18 / Chapter 1.4.3.1 --- Vascular endothelial growth factor (VEGF) --- p.18 / Chapter 1.4.3.2 --- Aptamer as a reversible antagonists of coagulation factor IXa is another example to show the potential case of aptamers as therapeutic agents --- p.20 / Chapter 1.4.4 --- Problem faced by aptamer --- p.21 / Chapter 1.4.4.1 --- Stability --- p.21 / Chapter 1.4.4.2 --- Clearance from blood --- p.22 / Chapter 1.5 --- Comparison between aptamer and antibody --- p.24 / Chapter 1.5.1 --- General comparison between aptamer and antibody --- p.24 / Chapter 1.5.1.1 --- Diversity --- p.24 / Chapter 1.5.2 --- Specificity --- p.26 / Chapter 1.5.3 --- Disadvantages of antibody --- p.26 / Chapter 1.5.4 --- Advantages of aptamer --- p.27 / Chapter 1.6 --- Project Objectives --- p.29 / Chapter Chapter 2. --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Chemicals --- p.32 / Chapter 2.1.2 --- Buffers --- p.36 / Chapter 2.1.2.1 --- Buffers commonly used --- p.37 / Chapter 2.1.2.2 --- Reagents for molecular work --- p.37 / Chapter 2.1.3 --- Bacterial Culture --- p.38 / Chapter 2.1.4 --- Culture of cell --- p.38 / Chapter 2.1.4.1 --- "TNF-α Sensitive Cell Line, L929" --- p.38 / Chapter 2.1.4.2 --- Medium for cell culture --- p.38 / Chapter 2.1.5 --- Reagent for Western blotting --- p.39 / Chapter 2.1.5.1 --- Protein extraction --- p.39 / Chapter 2.1.5.2 --- SDS-PAGE --- p.40 / Chapter 2.1.5.3 --- Electro-blotting --- p.41 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Conjugation of protein to solid support --- p.42 / Chapter 2.2.1.1 --- Conjugation of protein on PVDF membrane --- p.42 / Chapter 2.2.4.2 --- Conjugation of protein on Sepharose --- p.42 / Chapter 2.2.4.3 --- Conjugation of protein on magnetic bead --- p.42 / Chapter 2.2.2 --- SELEX --- p.43 / Chapter 2.2.2.1 --- Selection --- p.43 / Chapter 2.2.2.2 --- Photo-selection --- p.44 / Chapter 2.2.2.3 --- PCR --- p.45 / Chapter 2.2.3 --- Separation of oligonucleotides --- p.46 / Chapter 2.2.3.1 --- Separate short length double-stranded oligonucleotides by using polyacrylamide gel --- p.46 / Chapter 2.2.3.2 --- Separate short length single-stranded oligonucleotides by using denaturing polyacrylamide gel --- p.47 / Chapter 2.2.3.3 --- Extract the DNA from polyacrylamide gel --- p.48 / Chapter 2.2.3.4 --- Estimate the amount of DNA in solution after extraction --- p.49 / Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.49 / Chapter 2.2.4 --- Cloning of selected polyclonal aptamer --- p.50 / Chapter 2.2.4.1 --- Restriction cutting --- p.50 / Chapter 2.2.4.2 --- Ligation --- p.50 / Chapter 2.2.4.3 --- Preparation of the competent cells --- p.50 / Chapter 2.2.4.4 --- Transformation of plasmid into competent cell --- p.51 / Chapter 2.2.4.5 --- Plasmid extraction from bacterial culture --- p.51 / Chapter 2.2.5 --- Cell culture --- p.52 / Chapter 2.2.5.1 --- Cell culture of L929 --- p.52 / Chapter 2.2.5.2 --- Preservation of cells --- p.52 / Chapter 2.2.5.3 --- Treatment with TNF-α --- p.53 / Chapter 2.2.5.4 --- Fixation of cells --- p.53 / Chapter 2.2.6 --- Western blotting analysis --- p.54 / Chapter 2.2.6.1 --- Preparation of proteins from cells --- p.54 / Chapter 2.2.6.2 --- SDS polyacrylamide gel electrophoresis (SDS-PAGE) --- p.54 / Chapter 2.2.6.3 --- Electroblotting of protein --- p.55 / Chapter 2.2.6.4 --- Probing antibodies or aptamers for proteins --- p.55 / Chapter 2.2.6.5 --- Enhanced chemiluminescence (ECL) Assay --- p.56 / Chapter Chapter 3. --- Results --- p.57 / Chapter 3.1 --- Selection of aptamer against cytochrome c dotted on membrane with counter selection against BSA on membrane --- p.58 / Chapter 3.1.1 --- Selection process --- p.58 / Chapter 3.1.1.1 --- PCR cycles --- p.59 / Chapter 3.1.1.2 --- Polyclonal aptamer --- p.61 / Chapter 3.1.1.3 --- Monoclonal aptamer Contents --- p.63 / Chapter 3.1.2 --- Binding test of cy-1 to cy-4 to cytochrome c --- p.65 / Chapter 3.1.3 --- Binding of cy-3 to the cytochrome c dotted on PVDF membrane --- p.67 / Chapter 3.1.4 --- Test the binding of cy-3 with cytochrome c by ELISA --- p.68 / Chapter 3.1.5 --- Competitive binding between monoclonal aptamer cy-3 and anti-cytochrome c antibody --- p.70 / Chapter 3.1.6 --- Western blotting of pure cytochrome c by cy-3 --- p.71 / Chapter 3.1.7 --- Western blotting of pure cytochrome c from different species --- p.73 / Chapter 3.1.8 --- Cell lysate SDS-PAGE labeled with cy-3 --- p.75 / Chapter 3.1.9 --- Cell lysate labeled with cy-1 to cy-9 after SDS-PAGE --- p.77 / Chapter 3.2 --- Selection of cytochrome c-specific aptamer with counter selection against cytosolic protein --- p.79 / Chapter 3.2.1 --- Selection of aptamer against cytochrome c with counter selection against cytosolic cell lysate --- p.79 / Chapter 3.2.2 --- Selection of aptamer against cytochrome c by fixed cell followed by cytochrome c elution --- p.82 / Chapter 3.2.3 --- Selection of aptamer from cytochrome c band --- p.84 / Chapter 3.3 --- Primers Testing --- p.86 / Chapter 3.3.1 --- Cell lysate labeled with primers after SDS-PAGE --- p.86 / Chapter 3.3.2 --- Cell lysate labeled with cy-3 without primers --- p.87 / Chapter 3.3.3 --- Test the effect of sense oligonucleotide --- p.89 / Chapter 3.3.4 --- Sequence of monoclonal aptamer --- p.90 / Chapter 3.3.5 --- Cell lysate labeled with aptamers without primer ends --- p.92 / Chapter 3.3.6 --- Test of the aptamers after mutations --- p.93 / Chapter 3.3.7 --- Test for other biotinylated primers --- p.96 / Chapter 3.4 --- Elimination of non-specific binding --- p.98 / Chapter 3.4.1 --- Different types of cell lysate --- p.98 / Chapter 3.4.2 --- Heating effect on the non-specific binding --- p.99 / Chapter 3.4.3 --- Using milk as a blocking agent --- p.101 / Chapter 3.4.3.1 --- Milk blocked membrane --- p.101 / Chapter 3.4.3.2 --- Milk prevented the binding of aptamer to cytochrome c --- p.102 / Chapter 3.4.3.3 --- Cell lysate labeled with cy-3 after SDS-PAGE by using milk as blocking agent --- p.104 / Chapter 3.4.3.4 --- Aptamer selection against cytochrome c in the presence of milk --- p.105 / Chapter 3.4.4 --- Using DNA as a Blocking agent --- p.107 / Chapter 3.4.4.1 --- DNA blocked the non-specific binding --- p.107 / Chapter 3.4.4.2 --- Cell lysate labeled with cy-3 after SDS-PAGE by using DNA as blocking agent --- p.109 / Chapter 3.4.4.3 --- Selection against cytochrome c blocked by DNA --- p.110 / Chapter 3.4.4.4 --- "Labeling of cell lysate treated with DNase, RNase or both after SDS-PAGE" --- p.112 / Chapter 3.5 --- Photo-SELEX --- p.114 / Chapter 3.5.1 --- Selection process --- p.114 / Chapter 3.5.2 --- Cell lysate labeled with photo-aptamer --- p.116 / Chapter 3.5.3 --- Testing by immunoprecipitation --- p.118 / Chapter 3.6 --- Application --- p.120 / Chapter 3.6.1 --- Detection of the cytochrome c in cytosolic proteins after treatment of TNF-α --- p.120 / Chapter 3.6.2 --- Detection of the cytochrome c in total cell lysate after treatment of TNF-α --- p.123 / Chapter 3.6.3 --- Detection of cytochrome c in different cellular compartments after treatment of TNF-α --- p.125 / Chapter Chapter 4. --- Discussion --- p.130 / Chapter 4.1 --- General information --- p.131 / Chapter 4.1.1 --- The pool of oligonucleotide --- p.131 / Chapter 4.1.2 --- Design of oligonucleotides --- p.131 / Chapter 4.1.3 --- SELEX --- p.133 / Chapter 4.1.3.1 --- Buffer condition of selection --- p.133 / Chapter 4.1.3.2 --- Binding equilibrium --- p.134 / Chapter 4.1.3.3 --- Prevalence of matrix-binding species --- p.134 / Chapter 4.2 --- Selection --- p.135 / Chapter 4.2.1 --- Cycle numbers of PCR --- p.135 / Chapter 4.3 --- Assay of aptamers selected --- p.137 / Chapter 4.3.1 --- The use of biotin-streptavidin for recognition --- p.137 / Chapter 4.3.2 --- Polyclonal aptamers --- p.137 / Chapter 4.3.3 --- Monoclonal aptamer --- p.137 / Chapter 4.3.4 --- Cy-3 shows the highest affinity to cytochrome c --- p.138 / Chapter 4.3.5 --- The presence of non-specific binding --- p.138 / Chapter 4.4 --- Counter selection against cell lysate --- p.140 / Chapter 4.5 --- Primer testing --- p.143 / Chapter 4.6 --- Sequences and secondary structures of monoclonal aptamers --- p.145 / Chapter 4.7 --- Elimination of non-specific binding Contents --- p.147 / Chapter 4.7.1 --- Non-specific binding may be mediated by sequence-independent recognition --- p.147 / Chapter 4.7.2 --- Elimination of non-specific binding by milk --- p.147 / Chapter 4.7.3 --- Eliminate the non-specific binding by using DNA --- p.149 / Chapter 4.8 --- Photo-aptamer --- p.151 / Chapter 4.9 --- Application of the monoclonal aptamer cy-3 --- p.153 / Chapter 4.9.1 --- Aptamer can label cytochrome c as antibody does --- p.153 / Chapter 4.10 --- Conclusion I --- p.158 / Chapter 4.11 --- Conclusion II --- p.159 / Chapter Chapter 5 --- References --- p.161

Choy, Man Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-123). / Abstracts in English and Chinese. / Title Page --- p.1 / Abstract (English) --- p.2 / Abstract (Chinese) --- p.3 / Acknowledgement --- p.4 / Declaration --- p.5 / Table of Content --- p.6 / Lists of Figures --- p.9 / Lists of Tables --- p.12 / List of Schemes --- p.13 / Chapter Chapter One --- Introduction / Historical perspective and overview of tandem mass spectrometry for structural biochemistry --- p.14 / Electrospray ionization (ESI) --- p.15 / Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) --- p.18 / Chapter 1.3.1 --- History of FTICR --- p.18 / Chapter 1.3.2 --- Theory of FTICR --- p.21 / Chapter 1.4 --- Sequencing of DNA fragments --- p.26 / Chapter 1.4.1 --- Conventional and mass spectrometric sequencing techniques --- p.26 / Chapter 1.4.2 --- Fragment-ion nomenclature --- p.27 / Chapter 1.4.3 --- Tandem mass spectrometry of oligonucleotide ions --- p.29 / Chapter 1.4.4 --- Electron capture dissociation of oligonucleotide ions --- p.31 / Chapter 1.5 --- Outline of the present work --- p.32 / Chapter Chapter Two --- Instrument and Experimental / Chapter 2.1 --- Instrumentation --- p.35 / Chapter 2.1.1 --- Fourier-transform ion cyclotron resonance mass spectrometer --- p.35 / Chapter 2.1.2 --- Vacuum system --- p.35 / Chapter 2.1.3 --- Nanospray ion source --- p.39 / Chapter 2.1.4 --- Ion Transfer system --- p.41 / Chapter 2.1.5 --- Infinity cell --- p.43 / Chapter 2.1.6 --- Electron emission source --- p.44 / Chapter 2.2 --- Experimental section --- p.47 / Chapter 2.2.1 --- Simple acquisition pulse program --- p.47 / Chapter 2.2.2 --- ECD pulse program --- p.49 / Chapter Chapter Three --- Production of Doubly-prontonated Oligonucleotide ions using Nanospray Ionization / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Experimental and instrumental section --- p.53 / Chapter 3.2.1 --- Materials --- p.53 / Chapter 3.2.2 --- Sample preparation --- p.53 / Chapter 3.2.3 --- Instrumentation --- p.54 / Chapter 3.3 --- Results and discussion --- p.54 / Chapter 3.3.1 --- Effect of the concentration of ammonium formate --- p.54 / Chapter 3.3.2 --- Effects of the anionic pair of the ammonium salts --- p.57 / Chapter 3.3.3 --- Effects of solvent composition --- p.64 / Chapter 3.3.4 --- Effects of analyte concentration --- p.66 / Chapter 3.4 --- Conclusion --- p.68 / Chapter Chapter Four --- Electron Capture Dissociation of Model Oligonucleotides / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Experimental and instrumental section --- p.70 / Chapter 4.2.1 --- Materials --- p.70 / Chapter 4.2.2 --- Sample preparation --- p.70 / Chapter 4.2.3 --- Instrumentation --- p.71 / Chapter 4.2.4 --- Method of calculations --- p.71 / Chapter 4.3 --- Results and discussion --- p.72 / Chapter 4.3.1 --- "ECD of d(CCCCC), d(CCAAC), d(CCTTC) and d(CCGGC)" --- p.72 / Chapter 4.3.1.1 --- General features --- p.72 / Chapter 4.3.1.2 --- Protonated nucleobases and nucleoside-like fragments --- p.73 / Chapter 4.3.1.3 --- Doubly-charged fragment ions --- p.79 / Chapter 4.3.2 --- Theoretical calculation of electron capture affinities of common functionalities in oligonucleotides --- p.80 / Chapter 4.3.3 --- Electron capture dissociation of C/T binary-based oligonucleotides --- p.81 / Chapter 4.3.3.1 --- "ECD of d(CTCTC), d(TCCCT) and d(CTTTC)" --- p.84 / Chapter 4.3.3.2 --- ECD of d(CCCCT) and d(TCCCC) --- p.84 / Chapter 4.3.4 --- Mechanistic implications --- p.89 / Chapter 4.4 --- Conclusion --- p.99 / Chapter Chapter Five --- Electron Capture Dissociation of a Series of G/T Binary Base of Oligonucleotides / Chapter 5.1 --- Introduction --- p.100 / Chapter 5.2 --- Experimental and instrumental section --- p.100 / Chapter 5.2.1 --- Materials --- p.100 / Chapter 5.2.2 --- Sample preparation --- p.100 / Chapter 5.2.3 --- Instrumentation --- p.101 / Chapter 5.3 --- Results and discussion --- p.101 / Chapter 5.3.1 --- Electron capture dissociation of d(GGGGG) --- p.101 / Chapter 5.3.2 --- Electron capture dissociation of G/T binary-based oligonucleotides --- p.104 / Chapter 5.3.2.1 --- "ECD of d(GTGTG), d(GTTTG) and d(TGGGT)" --- p.104 / Chapter 5.3.2.2 --- ECD of d(GGGGT) and d(TGGGG) --- p.107 / Chapter 5.3.3 --- Mechanistic implications --- p.110 / Chapter 5.4 --- Conclusion --- p.117 / Chapter Chapter Six --- Conclusion Remarks --- p.118 / References --- p.120 / Appendix A --- p.124 / Appendix B --- p.127

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 190 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 153-180).

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xiii, 165 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 157-165).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 210 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 154-185).