PART I: SEARCH DEOXYRIBOZYMES FOR WITH NEW TYPES OF SELF-CLEAVING ACTIVITY UNDER HYDROLYSIS PATHWAY CHAPTER Introduction Nature has spent more than billion years to perfect the many thousands of enzymes that are utilized in living cells. Yet this long and merciless process of evolution has given rise to enzymes based on only two molecular formats-protein and RNA. In opting to build biocatalysts, nature has made two excellent choices. Proteins exploit the different chemistries offered by their constituent 20 amino acids to form an incredible array of diverse structures and precisely configured active sites. So together with their ability to adopt a seemingly endless array of tertiary structures in sequencedirected fashion, proteins are well suited to serve as enzymes. The splendid catalytic potential of proteins is exemplified by enzymes, such as orotidine 5’-phosphate decarboxylase, which coverts substrate to product with a rate enhancement of 17 orders of magnitude over the corresponding uncatalyzed rate [1]. Although RNA’s role in modern biocatalysis might be an “accidental catalysts” from life’s early evolutionary history [2], catalytic RNAs (ribozymes) are certainly capable of generating impressive rate enhancements. For example, group I ribozymes catalyze RNA splicing with a rate enhancement of ~13 orders of magnitude. Only eight classes of naturally occurring RNA biocatalysts have been identified so far since the 1980s when Thomas Cech made the first surprising discovery that RNA molecules are capable of catalyzing reactions in the absence of any protein components [3]. In contrast to proteins, the diversity of chemical functional groups in RNA is severely limited, and this fact is widely considered as the most significant determinant that constrains both the structural and catalytic potentials of RNA. Then why is RNA provided with the catalytic activity of enzymes from evolution? The most important reason is that RNA is capable of bringing its lesser chemical repertoire to bear on catalysis by forming surprisingly intricate three-dimensional structures [4-6]. The versatilities of RNA in forming intricate and functional structures are due to the combined use of standard Watson-Crick base pairing, non-standard base pairing, and a variety of non-covalent contacts involving phosphates, ribose oxygen and cationic metals. A significant component for building complex RNA structures is the 2' hydroxyl of ribose, as the oxygen atom of this group can act both as a donor and as an acceptor for hydrogen bonding [7]. 1.1 Classes of Ribozymes Ribozymes, or catalytic RNAs, were first discovered in the laboratory of Tom Cech, at the University of Colorado, in 1982. They found that the ribosomal RNA precursor from Tetrahymena thermophila contained an intron, a nonencoding sequence that interrupts the gene, and was capable of excising itself, in vitro, without any protein or external energy source [8]. Shortly thereafter, Sidney Altman's group, at Yale University, showed that the RNA component of RNase P, M1 RNA, from Escherichia coli was likewise able to process tRNA precursors without any protein factors [9]. This seminal work ushered in a major research activity, and RNA catalysis was soon found to be widespread in nature, occurring in plants, bacteria, viruses, and lower eukaryotes. Seven naturally occurring classes of catalytic RNAs have been identified to date (hammerhead, hepatitis delta virus, hairpin, Neurospora Varkud satellite, group I introns, group II introns and ribonuclease P), all of which catalyze cleavage or ligation of the RNA backbone by transesterification or hydrolysis of phosphate groups. The hammerhead, hepatitis delta virus (HDV), hairpin, and Neurospora Varkud satellite (VS) ribozymes are small RNAs of 50–150 nucleotides that perform sitespecific self-cleavage [10–16]. The general mechanism of these self-cleavage reactions is similar to that of many protein ribonucleases in which a 2’-oxygen nucleophile attacks the adjacent phosphate in the RNA backbone, resulting in cleavage products with 2’, 3’-cyclic phosphate and 5’-hydroxyl termini. Found in viral, virusoid, or satellite RNA genomes, these small catalytic RNAs process the products of rolling circle replication into genome-length strands [17]. A 5' 5' O 5' O O Base O Base O Base O HO O - O P OH O O - P O O OO H O O H H O OH O P - O- Base O O- O O + Base HO H Base O O 3' OH 3' O OH 3' B 5' 5' 5' O O H Base O O Base O O O Base H O O OH P - O O- Nuc O H O- O Base O P O OH + OO Nuc O O- Base O OH 3' OH OH O- O OH OH P O Nuc O O 3' O Base OH 3' Figure 1-1. Two mechanistic classes of ribozyme phosphodiester cleavage. Circles represent divalent cations and several ways that they may participate in catalysis. Although most of the ions are shown participating in the transition state of the reaction, they may also contribute to ground-state stabilization and substrate binding (represented by M2, the square labeled 2). (A) Mechanism A (mA), observed in metal catalyzed RNA hydrolysis and RNA cleavage by the hammerhead, hairpin, hepatitis delta, Neurospora, and tRNA ribozymes. The attack of the 2’-OH group proceeds by an in-line SN2 nucleophilic displacement with inversion of the configuration at phosphorus [43]. The M2 square represents a divalent cation that promotes catalysis by the hammerhead ribozyme. In addition to coordinating the nonbridging phosphate oxygen, M2 may also stabilize the oxyanion nucleophile. (B) Mechanism B (mB) found in catalysis by ribonuclease P, group I and group II intron ribozymes. The exogenous nucleophile (Nuc), which can be water or hydroxyl functionality, attacks the phosphorus center by means of SN2 displacement [136].The square labeled represents M3, a divalent ion that promotes catalysis by the Tetrahymena ribozyme. Group I introns, group II introns and ribonuclease P (RNase P) are larger, more structurally complex ribozymes with several hundred nucleotides in length [18–20]. RNase P cleaves precursor RNA substrates at specific sites to generate functional 5’- termini [21], and group I and II introns catalyze two-step self-splicing reactions [22– 24]. In these large ribozymes, the nucleophile and the labile phosphate are located on different molecules or are greatly separated in sequence. Thus, the complex folds of these RNAs serve to orient the nucleophile and phosphate to ensure accurate cleavage or splicing. 1.1.1 Hammerhead ribozyme structure and catalysis The hammerhead ribozyme was the first self-cleaving RNA to be discovered [25, 26], the first ribozyme to be crystallized [27, 28], and has been the focus of more studies than any other catalytic RNAs. Even so, a complete understanding of its reaction mechanism still remains elusive [29-31]. Hammerhead and hairpin ribozymes are found in opposite strands of the same plant virus satellite RNAs, and they catalyze identical chemical reactions [32]. Nonetheless, the two ribozymes adopt different structures and have different biochemical features, including different pH and metalcation dependencies and different proficiencies for catalyzing RNA ligation [33]. A minimal hammerhead contains three base-paired helices (helices I, II, and III) around a core of conserved nucleotides. Two crystal structures of non-cleavable variants of the hammerhead ribozyme have been solved, one with a DNA substrate strand and the other with an RNA substrate containing a 2’O-methyl group at the cleavage site [34, 35]. Both showed the three helices are arranged in a Y shape, as predicted by fluorescence and native gel electrophoresis data [36, 37]. Stems II and III are essentially coaxial, while helix I lies at a sharp angle to helix II. Backbone distortions at the junction of helices II and III force nucleotide C17 to stack on stem I rather than on stem III, placing it in the active site pocket at the three-helix junction. The scissile phosphate on the 3’-side of C17 lies above a hairpin turn of the backbone formed by the C3-A6 sequence. This CUGA turn is strikingly similar to that found in the anti-codon loop of tRNA, which serves as a metal binding pocket [38]. Many experiments have been performed to determine the mechanism of hammerhead self-cleavage and the role of divalent cations in the reaction. Sulfur substitution for the scissile phosphate oxygens showed that there is inversion of configuration about the phosphate during the reaction, indicating that the reaction proceeds by in-line attack of the nucleophile [32, 39-40]. Further, in most of the experiments in which sulfur was substituted for the pro-Rp non-bridging oxygen of the scissile phosphate, the reduced activity was rescued by addition of a thiophilic metal ion, suggesting that a metal ion directly coordinates this oxygen in the transition state [40-45]. The reaction rate increases linearly with pH, indicating that the nucleophile is activated by a hydroxide ion [46]. Either a metal ion hydroxide deprotonates the 2’-hydroxyl directly, or a metal ion coordinated to the 2’-hydroxyl increases the acidity of the 2’oxygen, rendering it susceptible to attack by a hydroxide ion from solution [46, 47]. The ion coordinated to the pro-Rp phosphate oxygen could perform either of these functions. If the hammerhead uses a two-metal ion mechanism for self-cleavage, then an additional directly coordinated ion should stabilize the leaving group oxygen [48, 49]. While sulfur substitution for the leaving group oxygen has failed to identify such a metal ion, other observations support its existence [47, 50-51]. Thus, there is debate over the number of divalent cations directly involved in hammerhead catalysis. Strikingly, recent experiments demonstrate that the ribozyme can function in the complete absence of divalent cations at extremely high ionic strengths [52]. This result showed that divalent cations are not essential cofactors in the reaction, though at least one directly coordinated magnesium ion appears to be involved in catalysis under most conditions in vitro. 1.1.2 Hairpin ribozyme structure and catalysis Another catalytic RNAs domain found in pathogenic plant virus satellite RNAs is the hairpin motif. Similar to the other small ribozymes, hairpin catalysts cleave concatameric precursor molecules into mature satellite RNA during rolling-circle replication, giving rise to a 2’-3’-cyclophosphate and a free 5’-OH terminus. Depending on reaction conditions, the hairpin ribozyme may also favour RNA ligation over cleavage [53]. Up to now, three different hairpin ribozymes have so far been found in nature, of which the one from satellite RNA associated with tobacco ring spot virus is the best characterized [54, 55]. The other two hairpin ribozymes, isolated from different satellite viruses, showed the sequence variations that preserve the overall structure of the molecules [56]. In naturally occurring hairpin ribozymes, the catalytic entity is a part of a four-helix junction. A minimal catalytic motif, containing approximately 50 nucleotides, has been identified that can be used for metal-ion dependent cleavage reactions in trans. It consists of two domains, each of them harbouring two helical regions separated by an internal loop, connected by a hinge region. One of these domains results from the association of 14 nucleotides of a substrate RNA with the ribozyme via base-pairing. In recent work, crystal structures of hairpin ribozymes bound to a modified, uncleavable substrate molecule, a transition state mimic, and a product complex have been obtained [57,58]. These structures and related biochemical data showed that the active site contains no tightly bound, well ordered metal ions at the site of cleavage. Catalytic activity of the hairpin ribozyme thus results from distortion and precise orientation of the substrate RNA and general acid base catalysis by nucleotides in neighborhood without involvement of metal ions in catalysis [55]. The requirement for metal ions may be explained by a significant role in the folding process [59, 60]. It has also been shown that catalytic activity of the hairpin ribozyme can be supported by spermine, the major polyamine in eukaryotic cells [61]. 1.1.3 Hepatitis delta virus and Varkud Satellite ribozymes The hepatitis delta virus ribozyme was found in a satellite virus of hepatitis B virus, a major human pathogen [62]. Both the genomic and the antigenomic strands express cis-cleaving ribozymes of ~85 nucleotides that differ in sequence but fold into similar secondary structures. A crystal structure of the ribozyme has been determined [63], in which five helical regions are organized by two pseudoknot structures. There is strong evidence that the catalytic mechanism of the hepatitis delta virus ribozyme involves the action of a cytosine base within the catalytic centre as a general acid-base catalyst. The hepatitis delta ribozyme displays high resistance to denaturing agents like urea or formamide. The Varkud Satellite (VS) ribozyme is a 154 nucleotides long catalytic entity that is transcribed from a plasmid discovered in the mitochondria of certain strains of Neurospora [64]. The VS ribozyme is the largest of the known nucleolytic ribozymes and the only one for which there is no crystal structure available to date. The global structure has been determined by solution methods, particularly Fluorescence Resonance Energy Transfer (FRET), which revealed a formal H shape of the five helical segments [65]. 1.1.4 General properties of introns Introns, or intervening sequences (IVS), are nonencoding sequences that interrupt the coding, exon, sequences. These introns must be removed, at the RNA level, in order for the gene to be expressed functionally. There are five major categories of introns and splicing mechanisms. These consist of nuclear tRNA introns [66], archaeal introns [67], nuclear mRNA introns [68], and the group I and group II introns [69, 70]. Of these introns, some members of the group I and group II are clearly capable of catalyzing their own excision, in vitro, in an RNA-catalyzed fashion. 1.1.4.1 Group I introns Group I introns are found in the nucleus and organelles of eukaryotes, as well as some prokaryotes and bacteriophages. These introns are composed of a strictly conserved core region essential for catalysis and moderately conserved peripheral regions that enhance their catalytic activity [71-74]. The mechanism of the catalytic reaction with group I introns involves excision from precursor RNAs through a two-step splicing reaction. A prerequisite for splicing is the binding of an exogenous guanosine (exoG) cofactor to a pocket in the catalytic core of the intron, referred to as G binding site. During the first step of splicing, the cofactor attacks the 5’-splice site (SS) and attaches to the intron, resulting in the release of the upstream exon. The exoG leaves the G-binding site and is replaced by the last nucleotide of the intron, which is always a guanosine 10 [41] Freudenreich, C. H.; & Kreuzer, K. N. EMBO J. 1993, 12, 2085. [42] Anderson, A. H.; Sorensen, B. S.; Christiansen, K.; Svejstrup. J. Q.; Lund, K.; Westergaard, O. J. Biol. Chem. 1991, 266, 9203 [43] Sabourin, M.; Osheroff, N. Nucleic Acids Research 2000, 28, 1947. [44] Bigioni, M.; Zunino, F.; Tinelli, S.; Austin, C. A.; Willmore, E.; Capranico, G. Biochemistry 1996, 35, 153. [45] Satoko Y.; Takuya U.; Yoshiharu I.; Kinichiro M.; Kinitsuna W.; Ichiro H. Nucleic Acids Res. 1994, 22, 2217. [46] Friedberg, E. C.; Walker, G. C.; Siede, W. 1995, DNA Repair and Mutagenesis, American Society for Microbiology, Washington, D. C. [47] Hande, K. R. Biochim. Biophys. Acta. 1998, 1400, 173. [48] Swift, L. P.; Rephaeli, A.; Nudelman, A.; Phillips, Don R.; Cutts, S. M. Cancer Research 2006, 66, 4863. 187 CHAPTER Materials and Methods 6.1 Materials 6.1.1 Oligodeoxyribonucleotide All oligodeoxyribonucleotides used in these studies were purchased from SigmaProligo and 1st Base Pte. Ltd. 6.1.2 Enzymes, chemicals and equipments Product (s) Manufacturer, Location T4 polynucieotide kinase New England Biolabs T4 DNA ligase New England Biolabs Human topoisomerase I Topogen Human topoisomerase II Topogen T7 exonuclease New England Biolabs pBR322 DNA New England Biolabs HCl, KCl, MgCl2, NaCl, NaOH Sigma Methylene blue Sigma Agarose Invitrogen γ-32P ATP Amersham PhosphorImager (Typhoon 8600) Amersham Gel Documentation System Syngnene, Cambridge, UK pH meter VWR, Singapore X-ray films Fuji, Singapore 188 X-ray development solution Kodak, Singapore Eppendorf tubes, pipettes tips, Greiner, Singapore NAP-25 GE, Healthcare Most all the chemicals used in this study were listed above otherwise were obtained from Sigma-Aldrich with analytical grade or even better quality. 6.1.3 Buffers and solutions All solutions were prepared using deionized water (Milipore MW-Q Water System, using osmotically purified H2O as source water) and highest purity reagents available Denaturing loading buffer: 7-8 M urea, or 98% formamide; 0.15% Xylene Cyanol; 0.15 % Bromophenol Blue; 18mM EDTA 1X T4 Polynucleotide Kinase Reaction Buffer: 70 mM Tris-HCl; 10 mM MgCl2 ; mM Dithiothreitol; pH 7.6, 25°C 1X T4 DNA Ligase Reaction Buffer: 50 mM Tris-HCl;10 mM MgCl2; mM ATP; 10 mM Dithiothreitol; pH 7.5, 25°C 1X T7 Exonuclease Reaction Buffer: 20 mM Tris-acetate; 50 mM Potassium Acetate; 10 mM Magnesium Acetate; mM Dithiothreitol; pH 7.9, 25°C 1X Human Topoisomerase I Reaction Buffer: 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, mM PMSF and 1.0 mM mercaptoethanol 1X Human Topoisomerase II Reaction Buffer: 50 mM Tris-Cl, pH 8.0, 120 mM KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol 6.2 Methods 6.2.1 Radioactive labeling DNA at the 5'–end with 32P 189 The T4 polynucleotide kinase (T4 PNK) from E. coli phage T4 was used to either transfer the radioactive terminal phosphate group of γ32P-ATP or the non-radioactive γ−phosphate of ATP to the free 5’-OH group of nucleic acids. The reaction mixture was prepared by adding the components in the order as they are listed below and incubated at 37°C for to hrs. Total volume: 20 μl: Final concentration 10 x T4 PNK buffer (forward reaction) μl 1x Deionized water 10 μl DNA purified by denaturing PAGE μl (100 μM ) 10 μM γ 32P ATP (3000 Ci/mmol, 10 μCi/μl, 3.3 μM) μl 0.66 μM 100 U / μl T4 PNK μl 10 U/μl After incubation, μl of loading buffer was added and the labelled DNA was purified by denaturing PAGE, eluted by diffusion and concentrated by NAP 25 columns. The purified DNA was dissolved in 100 μl of DNase-free water and the overall yield of labelled DNA determined by measuring with a Geiger counter. 6.2.2 Polyacrylamide gel electrophoresis (PAGE) Polyacrylamide gels are generated by crosslinking polymers of acrylamide with the co-monomer bisacrylamide. The covalent crosslinking reaction is catalyzed by TEMED, a tertiary amide, and initiated by ammonium persulfate (APS). Varying the concentration of acrylamide and bis-acrylamide can generate a wide range of pore sizes. 190 When molecules are placed in an electric field, they will migrate to the respective electrode with a velocity (electrophoretic mobility) proportional to the field strength and the net charge of the molecule. Polynucleotide chains of DNA or RNA are polyanions and migrate to the positive electrode (anode) when placed in an electric field. Since nucleic acid molecules migrate to the anode through the pores of a matrix of polyacrylamide or agarose, their velocity is significantly influenced by the size of both the molecule and the pores. Therefore, in a gel, larger molecules show a decreased electrophoretic mobility compared to smaller molecules with the same charge density. Generally, optimal electrophoresis conditions are often determined empirically for each situation, taking into consideration fragment length, required resolution and available time. 6.2.3 Elution by diffusion Elution by diffusion was applied to elute RNA molecules or DNA oligonucleotides from denaturing polyacrylamide gels. The DNA / RNA band of interest, detected by UV shadowing or using a phosphoimager (radiolabelled RNA / DNA), was excised and incubated overnight in 5-8 volumes of elution buffer at 4°C. The eluate was concentrated with NAP 25 column and dissolved in sterile RNase / DNase free water. Different elution buffers were used depending on the application. 6.2.4 NAP gel filtration NAP columns sold by Pharmacia are filled with Sephadex G 25 or G 50, a gel filtration matrix. They are used to remove salts and mononucleotides from nucleic acid solutions. By this method up to 97 % RNA / DNA can be recovered while more 191 than 90 % of the low molecular weight impurities are removed. In this work, NAP 25 columns were used. The columns were equilibrated with RNase free water. An exact volume (as defined by the supplier) of the nucleic acid solution was loaded onto the column and entered the gel matrix by gravity flow. Elution of the nucleic acids was accomplished by adding another precisely defined volume of RNase free water. For details, refer to the respective instruction manuals. 6.2.5 Ethanol precipitation of DNA Ethanol precipitation is the most straightforward method to concentrate nucleic acids from aqueous solutions. In 70 % ethanol the nucleic acids become insoluble and precipitate, while most salts will remain predominantly in solution and can thus be removed. Therefore, ethanol precipitation is also very useful when changes in salt conditions are required. When short RNA/DNA molecules (< 20 nucleotides in length) or very small amounts of RNA/DNA had to be precipitated, a better recovery was obtained if glycogen (20 to 40 μg) was used as carrier during the precipitation. For precipitation, to volume of RNA/DNA solution, 1/10 volume of M Sodium Acetate pH for precipitating unmodified RNA/DNA was added. After addition of 2.2 or 2.7 volumes of absolute ethanol, respectively, the sample was thoroughly mixed. The sample was stored at –20°C for at least h and then centrifuged at 15 000 x g and 4°C for h. The supernatant was removed with a sterile tip and the pellet eventually washed with 70 % ethanol, if a high salt concentration was present in the initial RNA/DNA solution, and centrifuged for another 15 minutes at 15 000 x g and 4°C. Finally, the supernatant was removed completely and the pellet dried at room temperature. The required time for drying the pellet depended on pellet size. When 192 drying was complete (pellet became transparent), the pellet was resuspended in the desired volume of sterile double distilled water or appropriate buffer. 6.2.6 Ligation of DNA fragments DNA ligation is used in order to link DNA fragments end to end using either complementary overhangs (‘sticky’ ends) or blunt ends. In this work the method was used to construct circular dumbbell-shaped DNA. The enzyme used to catalyse the ligation reaction was T4 DNA ligase from E coli phage T4. This enzyme covalently links the DNA strands by generating a phosphodiester bond between 3’-hydroxy and 5’-phosphate groups. The reaction mix was prepared by adding the components in the order they are listed below and incubated at 25 °C for to h. DNA ligation reaction, 20 μl: DNA fragments 0.4 μg / μl or 1.1 μM final concentration 1.8 to μl 0.1 to 0.33 μM 10 x T4 ligase buffer μl 1x T4 DNA ligase 10 U / μl μl U / μl Deionized water top up to 20 μl 6.2.7 Detection methods of DNA 6.2.7.1 Autoradiography 32 P radiolabelled DNA was detected with the help of a phosphorimaging analyser. The gel was wrapped with plastic foil and an image plate was exposed to it. The exposition time depended on the amount of radioactivity loaded on the gel (e.g. for a labelling reaction; overnight to resolve kinetic experiments). Then the image 193 plate was scanned with the help of a phosphoimager. The obtained image was analysed and quantified using the program Imagequant. 6.2.7.2 Methylene blue staining Methylene blue is a dye that binds to DNA via electrostatic interactions. And it has been used for quantitative or qualitative examination of RNA and DNA. Run the gel normally and then place in a 0.002% methylene blue (w/v, Sigma M-4159) solution in 0.1X TAE (0.004M Tris 0.0001 M EDTA) for 1-4 h at room temp (22°C) or overnight at 4°C. Diffusion of the DNA does not seem to be a problem for fragments as small as 100 bp. 6.2.8 Self-cleavage assays of deoxyribozymes To assess the DNA cleavage activity of self-cleaving molecules, radiolabeled precursor DNA was prepared by enzymatically tagging the 5’ terminus of synthetic DNAs in a reaction containing [γ-32P] ATP and T4 polynucleotide kinase, which was incubated at 37oC for 1hr. The resulting 5’-32P-labeled DNA was isolated by denaturing PAGE and recovered from the gel matrix by soaking in 10mM Tris·HCl (pH 7.5). The recovered DNA was concentrated NAP 25 columns and resuspended in deionized water. Self-cleavage assays using trace amounts of radiolabeled precursor DNA (~10-100 nM) was next incubated at 20 0C in the presence of mM NaCl for 12 hours followed by addition of KCl (final concentration mM), which was then kept at the same temperature for additional 12 hours. The self-cleavage reactions of radiolabeled precursor DNA were initiated next by adding MgCl2 to the mixture, which was further kept at 34 oC for a different period of time. Cleavage products were separated by denaturing PAGE and imaged by autoradiography or by phosphorImager 194 (Molecular Dynamics), and product yields were determined by quantitation (IMAGEQUANT) of the corresponding precursor and product bands. 6.2.9 pH dependency of the self-cleavage reaction Individual 5’-32P labeled DNAs (4B1-T) were dissolved in incubation buffers with pH values ranging from to prior to the cleavage step. After incubation for 48hrs, 10 mM MgCl2 (final concentration) was added to initiate the cleavage reaction. Aliquots were removed at various times, and the cleavage reactions were analyzed a 20% denaturing PAGE gel. 6.2.10 Alkali-ion dependency of the self-cleavage reaction The metal ion specificity was determined by monitoring the ability of the DNA enzyme to undergo self-cleavage at pH 7.4 by changing different ions or concentration, as demonstrated by the presence of cleavage product on a 20% denaturing PAGE gel. 6.2.11 Magnesium dependence of the self-cleavage reaction Individual 5’-32P labeled DNAs (4B1-T) were dissolved in cleavage buffer with MgCl2 concentrations ranging from nM to 10 mM. Aliquots were removed at various times, the cleavage reactions were analyzed a 20% denaturing PAGE gel. 6.2.12 Measurement of self-cleavage rate constant To characterize the DNA cleavage activity, 5’-end radiolabeled precursor DNA was incubated at 20 oC in the presence of 5mM NaCl for 12 hours followed by addition of KCl (final concentration mM), which was then kept at the same temperature for additional 12 hours. The self-cleavage reactions of 4B1-T were initiated next by 195 adding MgCl2 to the mixture, which was further kept at 34 oC for a different period of time. The kinetics of cleavage was followed by removing and mixing 10-μL aliquots with μL of formamide-dye mixture containing 50 mM EDTA to quench the reaction. The precursor and product were separated by gel electrophoresis under denaturing condition (20% polyacrylamide gel containing M urea, 0.05 M Tris-borate, pH 8.3, and 0.5 mM EDTA). The reaction products from the kinetic experiments were resolved by denaturing PAGE (4.2.1), an image plate was exposed to the polyacrylamide gel for at least h, scanned and digitized using a Typhoon 8600 phosphorimage analyzer and the software IMAGEQUANT. Images were imported into the program Molecular Dynamics and quantification boxes were positioned around bands representing substrate or uncleaved complex and product or cleaved complex. The data report, containing the values corresponding to the intensity of marked bands, was imported into the curve fitting software (Origin 6.0) and the fraction of product corresponding to each time point was calculated: F product = I product / (I product + I substrate) where I product is the band intensity corresponding to product or cleaved complex and I substrate is the band intensity corresponding to substrate or uncleaved complex. The fraction of product (Fproduct) was corrected by subtracting the fraction of product calculated for the zero control (Fzero) Fzero = Fproduct when t = The corrected f product was represented as a function of time and the data were fitted to the equation: F product = F endpoint × (1 – e - kobs× t) 196 where Fendpoint represents the maximal fraction of substrate that can be cleaved, T is time and kobs is the observed reaction velocity rate constant. 6.2.13 Hydrolysis of circular DNA with T7 exdonucleases T7 Exonuclease acts in the 5' to 3' direction, catalyzing the removal of 5' mononucleotides from duplex DNA. T7 Exonuclease is able to initiate nucleotide removal from the 5' termini or at gaps and nicks of double-stranded DNA. It will degrade both 5' phosphorylated or 5' dephosphorylated DNA. It has also been reported to degrade RNA and DNA from RNA/DNA hybrids in the 5' to 3' direction but is unable to degrade either double-stranded or single-stranded RNA. Circular oligodeoxyribonucleotides are known to resist the degradation by this enzyme due to the absence of open termini within their structures. In order to confirm the circular nature of its phosphate–sugar backbones, the newly formed product from our ligation reaction was purified via denaturing PAGE and then hydrolyzed by this exonuclease. A reaction mixture containing 1× T7 exonuclease reaction buffer (10 mM Tris (pH 8.1), 20mM NaCl, 5mM MgCl2, 5mM 2-mercaptoethanol, 10 U of T7 exonuclease and the identified circular product or linear precusor in a total volume of 20 μl was subsequently prepared and incubated at 37°C for 30 min. This reaction product was next analyzed through PAGE. 6.2.14 Agarose gel electrophoresis Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and 197 the warm gel solution then is poured into a mold (made by wrapping clear tape around and extending above the edges of an 18 cm X 18 cm glass plate), which is fitted with a well-forming comb. The percentage of agarose in the gel varied. Although 1% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than kb is required, a 0.7, 1.5, or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at room temperature, depending on the desired separation. 6.2.15 Inhibition assays on human topoisomerase I and II Topoisomerase I and II activity was measured by assessing relaxation of supercoiled DNA (scDNA). The reaction mixture (20 μL) contained: 1X reaction buffer, 400 ng/μL scDNA and unit of human topoisomerase I or II, oligonucleotides were used at different concentrations. inhibition effect of The mixtures were incubated for minutes and the topoisomerase-dependent relaxation of scDNA by oligonucleotides was estimated by agarose gel electrophoresis. Preincubation of topoisomerase I or II with oligonucletides was done using standard conditions without scDNA for minutes at 25oC. The relaxation reaction was started by addition of scDNA and the mixture was further incubated for 20 minutes at 37oC. Percentage of relaxation was defined as the ratio of band density of relaxed DNA over those of relaxed DNA plus supercoiled DNA [relaxed DNA/ (relaxed DNA + supercoil DNA)] while (100%- percentage of relaxation) was taken as the percent inhibition of 198 topoisomerases activity by oligonucleotides. The DNA bands were analyzed using Gel Documentation System (G:Box HR, Syngnene, Cambridge, UK) equipped with Gene Tools Software. 199 Xinming Li Department of Chemistry National University of Singapore, Singapore 117543 Tel: (65) 8178 3065; Fax: (65) 6791 1961 Email: G0403551@nus.edu.sg EDUCATION: Jan. 2005 – Present Ph. D. graduate student (Ph. D. degree to be awarded in January 2009) Nucleic acids Chemistry Department of Chemistry National University of Singapore Supervisor: Professor Tianhu Li Oct. 2003 – Jun. 2004 Visiting Student, Max Planck Institute for Polymer Research, Mainz, Germany Sep. 2000 – Jul. 2003 Graduate student (Master degree awarded in June 2003) Materials Science and Engineering Department of Materials Science and Engineering Beijing University of Chemical Technology, Beijing, China Supervisor: Professor Yang Zhang Sep. 1994 – Jul. 1998 Undergraduate student (Bachelor degree awarded in June 1998) Polymer Science and Engineering Department of Polymer Science and Engineering Beijing University of Chemical Technology, Beijing, China RESEARCH EXPERIENCE: Jan. 2005-Present Graduate student working in the field of nucleic acid chemistry Department of Chemistry, National University of Singapore, Singapore Oct. 2003-Jun. 2004 Visiting Student working on fabrication of DNA biosensor on thermo-responsive hydrogel monolayer. Max Planck Institute for Polymer Research, Mainz, Germany Sep. 2000-Jul. 2003 Graduate student working on construction of high mechanical performance composites with glass fiber reinforced phenolic resin. Deparment of Polymer Science and Engineering, Beijing University of Chemical Technology, Beijing, China Sep. 1994-Jul. 1998 Undergraduate student working on investigation on the influence of UV radiation on photodegradation processes of poly (vinyl chloride) with different average degrees of polymerization. Deparment of Polymer Science and Engineering, Beijing University of Chemical Technology, Beijing, China MAJOR ACCOMPLISHMENTS IN NUCLEIC ACIDS AND POLYMER CHEMISTRY: • Construction of DNA machines that conduct translational motion along linear paths • Construction of i-motif-containing DNA device that breaks Watson-Crick interactions • Developed unimolecularly circular G-quadruplexes and i-motif containing fluorecein tag to be used as single molecular probes • Design and synthesis of dumbbell-shaped circular oligonucleotides as human topoisomerase I inhibitors 200 • • • Developed gap-containing unimolecular oligonucleotides as human topoisomerase II inhibitors Discovered site specific self-cleavage reaction of externally looped Gquadruplexes (Deoxyribozymes) Discovered self-cleaving G-quadruplexes composed of prokaryotic and eukaryotic telomere repeats TEACHING EXPERIENCE: Aug. 2005–Apr. 2007: Teaching Assistant (Course: Advanced experiments in analytical and physical chemistry-CM3292, in charge of Gas Chromography, Cyclic Voltammetric and Gas Chromography-Mass Spectrometry) SKILLS: • Design of novel structures of G- quadruplex, i-motif and triple helix DNA for desired applications • DNA purification and analysis by polyacrylamide and agarose gel electrophorese • Proficient with radioactive labeling of DNA and oligonucleotides with 32P • Expert in methods and techniques required for examining DNA-cleavage by small molecules • Expert in operating PhosphorImager, Gel Documentation and Analysis System, CD spectrophotomer, UV-Vis Spectrophotometer, Fluorescence Spectrophotometer, Microplate Reader. PUBLICATIONS: [1] Xinming Li, Magdeline Tao Tao Ng, Yifan Wang, Tianyan Zhou, Sock Teng Chua, Weixing Yuan and Tianhu Li, “Site-specific self-cleavage of G-quadruplexes formed by human telemetric repeats” Bioorganic & Medicinal Chemistry Letters, 2008, 18, 5576. [2] Xinming Li, Magdeline Tao Tao Ng, Yifan Wang, Xiaoqian Liu and Tianhu Li, “DumbbellShaped Circular Oligonucleotides as Human Topoisomerase I Inhibitors”, Bioorganic & Medicinal Chemistry Letters, 2007, 17, 4967. [3] Yifan Wang, Xinming Li, Xiaoqian Liu, Tianhu Li, “An i-motif-containing DNA device that breaks certain forms of Watson–Crick interactions”, Chemical Communications, 2007, 42, 4369. [4] Xiaoqian Liu, Xinming Li, Tianyan Zhou, Yifan Wang, Magdeline Tao Tao Ng, Wei Xu and Tianhu Li, “Site specific self-cleavage of certain assemblies of Gquadruplex”, Chemical Communications, 2008, 3, 380. [5] Zhou, Tianyan, Wang, Yifan, Li, Xinming, Zhang, Qiang, Li, Tianhu, “Synthesis and Characterization of A Fluorescein-Labeled Circular G-Quadruplex”, Bulletin of the Chemical Society of Japan, 2006, 79, 1300. [6] Yifan Wang, Magdeline Tao Tao Ng, Tianyan Zhou, Xinming Li, Choon Hong Tan, Tianhu Li, “C3-Spacer-containing circular oligonucleotides as inhibitors of human topoisomerase I”, Bioorganic & Medicinal Chemistry Letters, 2008, 18, 3597. [7] Xiaolin Li, Xinming Li, Yang Zhang, “Study of Phenolic Resin Toughened by Grafting with NBR”, China Synthetic Rubber Industry, 2002, 25, 115. 201 [8] Xinming Li, Xiaolin Li, Daming Wu, “Synthesis of Resole/Montmorillonite Nanocomposite by Intercalation Polymerization”, China Synthetic Rubber Industry, 2002, 25, 173. [9] Xinming Li, Xiaolin Li, Zhiqiang Su, Yang Zhang, “Phenolic resin modified by copolymerization with nitrile butadiene rubber”, Thermosetting Resin, 2002, 17, 11. MANUSCRIPTS SUBMITTED AND IN PREPARATION: [1] Tianyan Zhou, Xinming Li, Yifan Wang, Magdeline Tao Tao Ng, Sock Teng Chua, Ngee Mien Quek, Zhaoqi Yang, Ji Luo, Weixing Yuan and Tianhu Li, “Synthesis and characterization of circular structures of i-motif tagged with fluoresceins” (Submitted to Bioconjugate Chemistry on October 23, 2008) [2] Xinming Li, Magdeline Tao Tao Ng, Yifan Wang, Tianyan Zhou, Sock Teng Chua, WeixinYuan and Tianhu Li, “Site-specific self-cleavage of G-quadruplexes formed by Oxytricha telemetric repeats” (Manuscript in preparation) CONFERENCE PAPERS/POSTER PRESENTATION: [1] The First International Meeting on G-Quadruplex DNA (Poster Presentation) held at James Graham Brown Cancer Center, Louisville, KY, USA (April 21-24, 2007) [2] International Symposium by Chinese Inorganic Chemists & International Symposium by Chinese Organic Chemists (Oral Presentation) held at Grand Copthorne Waterfront Hotel, Singapore (December 17-21, 2006) 202 [...]...(denoted as ωG) The second step is initiated by an attack by the 3’-end of the released exon on the 3’ SS, which results in ligation of the exons and release of the intron RNA Successful catalysis is dependent on the correct folding of the intron [7577] Group I intron splicing requires divalent metal ions for proper folding and catalysis [77] Mg2+ coordination to substrate oxygen atoms activates the nucleophile,... that runs on the outside of the guanine tetrad core 24 There is considerable variation in quadruplex structures, depending on the DNA sequence and the ionic conditions [121] The biological function of quadruplexes may well depend on the folded conformation that is adopted, especially if this involves interaction with specific proteins Such an effect has been suggested for the NHE element of the c-myc... selection process of deoxyribozymes: A population of single stranded DNA molecules (top) is chemically synthesized with regions of random DNA sequence (box) and regions of known DNA sequence (plain lines) used for example as PCR primer binding sites This population is subjected to the desired selective conditions (1) during which the portion of the population of different molecules that can perform the. .. will then accelerate the cleavage of the abasic DNA linkage In 2 mM Ca2+, the “10–28” enzyme catalyzes 19 depurination of a separate substrate oligonucleotide with a pseudo-first-order rate constant of 0.2 min–1 This corresponds to a rate enhancement for depurination of ~1 million fold over the corresponding uncatalyzed reaction The mechanism could be an SN1 type, where protonation at the N7 position of. .. stabilizes the scissile phosphate and stabilizes the developing charge on the leaving group The proton of the 2’-OH at the cleavage site is shared between the 2’ -and 3’oxygens in the transition state [78] 1.1.4.2 Group II introns Group II introns are found in low frequencies in the mitochondrial genomes of fungi, sporadically in the organellar genomes of algae They are numerous in the organellar genomes of. .. confirm that the occurrence of self-cleavage reaction is fully dependent on the proper conformation of G-quadruplex assembly, and to explore more catalytic potentials with these G-quadruplex assemblies, additional oligonucleotide sequences with different catalytic loop geometry are designed and tested for their catalytic activities Finally, a number of metal and non-metal ion cofactors such as Mg2+ and histidine... ion [120], and can adopt a variety of different folding patterns depending on the relative orientation of the strands and the position of the loops 23 Figure 1-6 The guanine tetrad motif and its hydrogen bonding scheme For intramolecular quadruplexes, the four G-tracts are separated by loops These are of various lengths and can be as short as a single nucleotide [121] The loops can be arranged in several... experimentally for this enzyme system 20 Figure 1-5 DNA cleavage reaction by depurination process (a) The proposed mechanism of the 10–28 deoxyribozyme involves protonation at the N7 position of guanine, which facilitates the departure of the base moiety and permits subsequent attack by a water molecule at the resulting C1’ carbocation of deoxyribose (b) Proposed secondary structure of the 10–28 N-glycosylase... acid/base catalysts in the form of metal hydroxides and it can also partially neutralize the negative charge density on the backbones of single strand DNA to promote its folding into complex shapes Histidine is chosen as a candidate cofactor because of the potiental for the imidazole side chain to function in both general acid and general base catalyses near neutral pH Histidine is one of the residues most... strictly requires the imidazole group of histidine for catalysis Furthermore, DNAzymes can overcome these functional limitations by utilizing divalent metal ions cofactors for catalysis [117-118] Metal ions would function in two ways First, they would partially neutralize the negative-charge density on the backbones of single-stranded DNA, and promote its folding into complex shapes Second, water molecules . step of splicing, the cofactor attacks the 5’-splice site (SS) and attaches to the intron, resulting in the release of the upstream exon. The exoG leaves the G-binding site and is replaced by the. plasmid discovered in the mitochondria of certain strains of Neurospora [64]. The VS ribozyme is the largest of the known nucleolytic ribozymes and the only one for which there is no crystal. functionally. There are five major categories of introns and splicing mechanisms. These consist of nuclear tRNA introns [66], archaeal introns [67], nuclear mRNA introns [68], and the group I and