47 research outputs found

    Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

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    A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1 min−1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA

    Recent advances in DNA catalysis

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    DNA catalysts as phosphatases and as phosphoserine lyases

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    Proteins and RNA are the only known biopolymers that have catalytic roles in nature, whereas DNA is primarily considered to store and transfer genetic information. However, artificial single-stranded DNA has been identified by in vitro selection to catalyze several chemical reactions and several of those are of biological relevance. For in vitro selection or directed evolution of proteins, direct amplification is not possible, and it essential to attach the genotype to the phenotype. For nucleic acids however, the functional biopolymer can be readily amplified. DNA has the advantage of being directly amplified by polymerases, whereas RNA requires an additional reverse transcription step. Moreover, DNA catalysts identified by in vitro selection processes have shown similar catalytic proficiency as RNA. DNA has added advantages of low cost of chemical synthesis and higher stability. Considering these factors combined, identification of artificial DNA catalysts (deoxyribozymes) for chemical reactions is a valuable endeavor with long-term implications. Protein post-translational modifications (PTMs) are highly important in biological processes involving cellular regulation. Additionally, PTMs serve as important intermediates or key motifs on natural products and bioactive peptides. The natural protein enzymes carrying out the essential modifications may have several shortcomings for biotechnological use. Identification of artificial DNA catalysts with ability to perform chemoselective post-translation chemical reaction would be highly useful in studying biological regulatory processes, performing synthesis and late-stage diversification of post-translationally modified peptides, as well as carrying out other important functions that natural proteins may not readily solve. My first effort was to identify DNA enzymes with peptide/protein phosphatase activity, more specifically dephosphorylation of peptide side chains. Without a catalyst, phosphomonoester hydrolysis reactions have exceedingly low spontaneous reaction rates. Nature utilizes proficient protein enzymes to perform this challenging reaction with great efficiency. Using a known DNA catalyst for the in vitro selection process, new DNA catalysts were identified with phosphatase activity. The phosphatase DNA catalysts exhibited multiple-turnover activity with phosphotyrosine-containing free peptides and were active even in the presence of externally added cell lysate or bovine serum albumin (BSA). Furthermore, the best DNA phosphatase functioned with a larger protein substrate. This established the fundamental ability of DNA to catalyze dephosphorylation of amino acid side chain residues. The study also suggested that phosphatase DNA catalysts could perform intracellular phosphatase activity. Hence, these deoxyribozymes were functionalized on gold nanoparticles and delivered inside live mammalian cells to investigate if they behave as functional protein analogues (or mimics) of recombinantly expressed Protein Tyrosine Phosphatase (PTP1B). Separately, efforts were directed towards the important goal of identifying sequence-selective phosphatase deoxyribozymes. Although three separate efforts were directed towards identifying sequence-selective phosphatases deoxyribozymes, we were unsuccessful in accomplishing this specific goal of selectivity in the context of peptide sequence discrimination. Dehydroalanine (Dha) is a non-proteinogenic electrophilic amino acid that serves as a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides and microcystins. DNA enzymes were identified to establish the fundamental catalytic ability to eliminate phosphate from phosphoserine (pSer) to form Dha, namely phosphoserine lyase activity. Furthermore, DhaDz1 was utilized to achieve chemo-enzymatic synthesis of a cyclic cystathionine-containing peptide. Based on this initial success, future efforts will be directed to achieve sequence-general phosphoserine and phosphotyrosine lyase activity. Separately, application of sequence-general lyases in the synthesis of complex lanthipeptides and enrichment of phosphopeptides/proteins in phosphoproteomics will be explored

    Kinase deoxyribozymes

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    Nature has developed the use of proteins and RNA as enzymes, while DNA is used for the storage and transfer of genetic information. Proteins and RNA are biopolymers that can fold into specific secondary and tertiary structures to enable catalysis. Considering the structural similarity to RNA, single-stranded DNA should also be able to form complex structures capable of catalyzing reactions. DNA catalysts have not been identified in nature, but in vitro selection has led to the identification of DNA catalysts for a variety of chemical reactions. Identification of new catalysts favors the use of DNA for multiple reasons. Amplification of functional DNA sequences is directly possible using natural polymerases, whereas amplification of RNA requires an additional reverse transcription step and amplification of proteins is not possible. The total number of possible sequences is smaller for nucleic acids (4n, where n is the number of residues) than for proteins (20n). Within this sequence space a large number of random nucleic acid sequences will fold into secondary and tertiary structures unlike proteins which require specific amino acid sequences to form complex structures. Therefore, in vitro selection experiments to identify DNA catalysts will cover a large portion of sequence space, and a large fraction of the covered space will contain structured DNA sequences with the potential to be catalytically active. The ease of synthesis and stability of DNA compared to RNA or proteins also provides an advantage for its use as a catalyst. Natural post-translational modifications (PTMs) are important in biological systems. PTMs modulate protein activity resulting in rapid changes to cellular processes. Studying the role of specific PTMs is often limited to the ability to generate site-specific post-translationally modified proteins of interest. Phosphorylation of amino acid side chains is an abundant natural PTM that is essential for cellular function. Protein kinases, which catalyze phosphorylation, are often motif specific. Engineering these natural kinases to change motif requirements is challenging and often results in decreased substrate specificity. To identify new catalysts for the site-specific phosphorylation of a desired protein the use of DNA as a catalytic biomolecule is advantageous because an initially random population of DNA sequences does not have substrate biases, and DNA is a large biopolymer with the possibility to interact specifically with the substrates. Both ribozymes and deoxyribozymes have been identified to catalyze the phosphorylation of oligonucleotides. However, previous efforts to identify kinase deoxyribozymes to catalyze the phosphorylation of amino acid side chains were unsuccessful because the -thiophosphoryl donor used was not stable in the selection conditions. As described herein, a new in vitro selection method was developed using a previously identified deoxyribozyme to separate the active deoxyribozymes from the inactive DNA sequences. This method led to the identification of the first kinase deoxyribozymes capable of phosphorylating tyrosine residues within a tethered peptide substrate using a bound 5′-triphosphorylated RNA oligonucleotide as the phosphoryl donor. Separate selection experiments were performed using 1 mM GTP as the phosphoryl donor. The identified DNA catalysts are able to phosphorylate tyrosine within a peptide substrate and require only low micromolar concentrations of GTP. Site-specific modification of proteins is often desired. Most deoxyribozymes identified to modify peptide substrates have been identified using peptide substrates containing the reactive residue flanked by alanine residues. Peptide sequences derived from natural proteins contain a variety of amino acid residues with diverse functional groups that could be a point of interaction between the peptide substrate and DNA catalyst. Selection experiments were performed with biologically derived peptide sequences to identify tyrosine kinase deoxyribozymes with the ability to phosphorylate peptides sequence-specifically. Of the three peptide substrates evaluated the use of one led to deoxyribozymes that are peptide motif-specific, the second peptide led to deoxyribozymes with partial peptide sequence-selectivity, and the third did not lead to the identification of deoxyribozymes. The identification of peptide motif-specific deoxyribozymes demonstrates that DNA catalysts can interact specifically with peptide substrates, and individual DNA enzymes can interact with the same peptide substrate in a different manner. The ability to phosphorylate substrates that are free in solution is desired. However, previously identified kinase deoxyribozymes are unable to phosphorylate untethered peptide substrates. Original efforts increased the length of the tether between the peptide substrate and DNA anchor to mimic a peptide free in solution. These selection experiments did not lead to deoxyribozymes, and further analysis of other deoxyribozymes with untethered peptide reactivity suggests the long tethers may interfere with catalysis. Further efforts have focused on the incorporation of hydrophobic modifications into the DNA catalysts to improve peptide binding. DNA aptamers containing hydrophobic modifications have improved protein binding. Increased binding affinity between the peptide substrate and DNA catalyst may enable untethered peptide reactivity. While initial efforts focused on tyrosine phosphorylation, serine phosphorylation is also abundant in nature. Serine kinase deoxyribozymes have been identified to phosphorylate serine within tightly tethered peptide substrates using 5′-triphosphorylated RNA as the phosphoryl donor. Subsequent efforts to increase the tether length or use ATP as the phosphoryl donor were unsuccessful. Efforts to improve DNA catalysts with the ability to phosphorylate serine include using biologically derived peptide sequences to increase interactions between the deoxyribozyme and peptide substrate, and incorporating catalytically participatory modifications into the DNA enzymes

    Molecular cloning and Biochemical characterization of pyridoxal 5’-phosphate dependent enzymes of unknown function

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    General introduction The main topic of this Ph.D. thesis is the characterization of gene products identified as PLP-dependent enzymes with unknown function. A complete and detailed description on function and evolution of PLP-dependent enzymes is provided in chapter 1. Chapter 2 describes the molecular cloning as well as the recombinant expression and the initial biochemical characterization of a threonine synthase homolog from mouse. Finally, in chapter 3 I describe the study of two recombinant human proteins homolog of alanine-glyoxylate amino transferase 2. The last part, Appendix, reports the evaluation of small RNA-cleaving DNAs (ribozymes), chemically modified by means of introduction of monomers of locked nucleic acids (LNA), in order to improve to cleavage of long and structured mRNAs.Introduzione generale Argomento principale di questa tesi di dottorato è la caratterizzazione di prodotti genici identificati come enzimi PLP-dipendenti, ma aventi funzione ignota. Nel capitolo 1 viene data una introduzione completa e dettagliata sull’evoluzione e le caratteristiche degli enzimi PLP-dipendenti. Nel capitolo 2 sono descritti il clonaggio, l’espressione in forma ricombinante e l’iniziale caratterizzazione biochimica, di una protiena di topo omologa della treonina sintasi microbica. Nel capitolo 3 è illustrato lo studio di due proteine umane, omologhe dell’alanina-gliossilato amino transferasi 2. Nell’ultima parte, l‘Appendice, sono descritti studi su piccoli DNA ad attività ribonucleasica (ribozimi) modificati attraverso l’introduzione di monomeri di locked nucleic acid (LNA), con lo scopo di migliorare il taglio di mRNA dotati di struttura secondaria

    DNA enzymes for tyrosine PEGylation and azido-adenylylation of peptide and protein substrates

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    Proteins and RNA are used as enzymes in nature, while DNA is used for the storage and transfer of genetic information. Proteins and RNA are biopolymers that can fold into complex secondary and tertiary structures to enable substrate binding and catalysis. Given the structural similarity to RNA, single-stranded DNA should also be able to function as enzymes. DNA enzymes, or deoxyribozymes, have not been found in nature, but in vitro selection has led to the identification of deoxyribozymes for a variety of reactions. De novo enzyme identification favors the use of nucleic acids over proteins for several reasons. First, nucleic acids can be amplified by natural enzymes, whereas proteins cannot be amplified in any way. Second, the number of possible sequences is smaller for nucleic acids (4n, where n is the length of the biopolymer) than for proteins (20n). Therefore, selection experiments for identifying nucleic acid enzymes will cover a larger fraction of total sequence space. Furthermore, within the sequence space evaluated, a large portion of nucleic acid sequences will fold into secondary and tertiary structures, whereas most random protein sequences are unlikely to fold into high-order structures. Considering nucleic acid enzymes, DNA has additional advantages over RNA because DNA can be directly amplified by polymerases whereas RNA requires an extra reverse transcription step. DNA is also cheaper and more stable compared to RNA. Post-translational modifications (PTMs) are essential for protein functions. The ability to site-specifically modify peptides and proteins will enable better understanding and applications of these biomolecules. The idea of using DNA enzymes for peptide and protein modification is very attractive, especially considering that DNA enzymes with site selectivity can be de novo identified without the requirement of a known enzyme as the starting point. PEGylation is an important artificial PTM for therapeutic peptides and proteins. PEGylation improves the pharmacokinetic properties of biopharmaceuticals by increasing circulation half time, reducing immunogenicity, increasing solubility, and suppressing aggregation. Peptide and protein PEGylation is most commonly achieved by solely chemical means. However, these chemical strategies generally lack site selectivity among different target sites in the substrates. Some chemical strategies also suffer from off-target reactivity, i.e., poor chemoselectivity. Enzymatic approaches for PEGylation have also been developed, yet their application is limited by the substrate specificities and the sequence selectivities of the natural enzymes used. In Chapter 2, DNA enzymes were identified for PEGylation of tyrosine in a DNA-tethered peptide substrate using a 5′-phosphorimidazolide-activated oligonucleotide-PEG conjugate (Imp-oligo-PEG5k) as the PEG donor. Two different approaches are described for the identification of DNA enzymes that are functional with untethered peptide substrates. The first approach is to increase the length of the tether between the peptide substrate and the DNA anchor for mimicking a peptide free in solution. The selection experiment using this approach did not lead to deoxyribozymes, and further analysis of other deoxyribozymes with untethered peptide reactivities suggests that the long tethers may interfere with catalysis. Thus, the first approach was discontinued. The second approach is to alternate the position of the tether between the peptide substrate and the DNA anchor. The rationale is that DNA enzymes are expected to perform catalysis without the requirement of any tether if the enzymes are identified from selection experiments with alternating tether positions. Ongoing efforts include selection experiments using the second approach and mixed-sequence peptide substrates to identify deoxyribozymes with untethered peptide reactivity. In Chapter 3, a two-step strategy is described for DNA-catalyzed peptide modification. In this strategy, a DNA enzyme first catalyzes the transfer of the 2′-azido-2′-deoxyadenosine 5′ monophosphoryl group (2′-Az-dAMP) from the analogous 5′-triphosphate (2′-Az-dATP) onto the tyrosine hydroxyl group (azido-adenylylation). Second, a particular modification of interest is attached to the azido group by copper-catalyzed azide-alkyne cycloaddition (CuAAC) using an alkyne-functionalized reagent. Eleven deoxyribozymes with azido-adenylylation activity are described in Chapter 3. One of the DNA enzymes is selective for the YPR sequence motif and is able to discriminate between tyrosine residues within a single peptide on the basis of sequence context. Another deoxyribozyme is peptide sequence-general, functions with free peptides, and allows their subsequent CuAAC labeling with moieties such as PEG and fluorescein. The use of azido-adenylylation deoxyribozymes is a versatile method for the synthesis of site-specifically modified peptides and proteins, since the azide group installed by the DNA enzyme can be used for any particular modification as long as the corresponding alkyne derivative is available. One of our long-term goals is DNA-catalyzed site-specific modification of protein substrates. In Chapter 4, two proteins, human annexin V and human TNF-related apoptosis-inducing ligand (TRAIL) 114–281, and a 36-mer peptide pancreatic polypeptide (PP) with an additional C-terminal cysteine were used as the substrates to evaluate two different approaches for identifying deoxyribozymes. The first approach is to directly use protein substrates during in vitro selection experiments. This approach requires the surviving deoxyribozymes to simultaneously adopt functions of both binding to the protein substrates and catalyzing the modification. However, the selection experiments using this approach did not lead to deoxyribozymes. The second approach is to decouple the binding and catalytic functions required for DNA-catalyzed protein modification. In this modular approach, the binding function is assigned to the predefined aptamer domain, which is placed adjacent to the initially random enzyme domain. The sequence of the enzyme domain will be subsequently identified through in vitro selection in the presence of the aptamer domain. Ongoing efforts are focused on the identification of DNA aptamers that bind to annexin V, TRAIL, and PP, with benzyl, naphthyl, and indolyl modifications. Once the DNA aptamers are identified, they will be used as the binding modules in selection experiments to identify DNA enzymes for protein modification

    DNA-Based Ligands for Use in Asymmetric Catalysis and Development of Metallo-(deoxy)Ribozymes

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    The fascinating way nature relies on biomolecules, mostly proteins and sometimes RNA, to carry out sophisticated chemical processes led to more and more efforts to use the concepts of biology for preparing efficient chiral catalysts. The “hybrid catalyst” approach that combines the steric information derived from a protein scaffold with the catalytic activity of transition metal complexes offers a resourceful means of developing semisynthetic metalloenzymes for enantioselective applications. Since the discovery of nucleic acids with enzyme-like functions, the catalytic potential of nucleic acids is being revealed by in vitro selection and evolution of novel ribozymes and DNAzymes. Nucleic acids, especially RNA, appear to be versatile catalysts capable of accelerating a broad range of reactions and exquisitely discriminating between chiral targets. However, while proteins dominated the construction of hybrid catalysts, the application of DNA and RNA in asymmetric catalysis has hardly been explored. This work aimed at exploring the chirality of nucleic acids and generating hybrid catalysts based on DNA and RNA. Towards the development of metallo-(deoxy)ribozymes assisted by combinatorial strategies (e.g., SELEX), a straightforward synthetic way of embedding transition metal complexes in nucleic acids folds was established. DNA sequences carrying mono- and bidentate phosphine ligands as well as P,N-ligands were successfully prepared starting from amino-modified oligonucleotide precursors. The optimized “convertible nucleoside” approach allowed the parallel, high-yielding synthesis of various alkylamino-DNA conjugates differing in length and structure of the spacer. Coupling of amino-oligonucleotides with PYRPHOS, BINAP and PHOX ligands equipped with a carboxyl group led to the incorporation of phosphine moieties at predetermined internal sites. Moreover, the stability of the DNA-tethered BINAP and PHOX was reasonably high, which makes them attractive candidates for the development of transition metal-containing oligonucleotides. To this end, systematic studies on the behavior of phosphine- and PHOX-metal complexes in aqueous medium - a prerequisite of nucleic acid catalysts - were carried out. Two model organometallic transformations were selected that were compatible with the structure and chemistry of nucleic acids. The rhodium(I)-catalyzed 1,4-addition of phenyl boronic acid to 2-cyclohexen-1-one and iridium(I)-catalyzed allylic amination of the branched phenyl allyl acetate, respectively, proceeded efficiently in the presence of phosphorus-based ligands, in aqueous medium, at room temperature and low catalyst concentration. For the first model reaction, the best conversion (80%) was achieved with the isolated [Rh(nbd)BINAP]BF4 complex, in 6:1 dioxane/water, and TEA additive. On the basis of these data, a suitable system for assessing the catalytic potential of the DNA-BINAP ligand was implemented. In the second chosen reaction the in situ formed Ir(I)-PHOX complexes (0.05-0.1 mM) gave rise to racemic, branched allylic amination products in good yields (33-75%), in 3:7 dioxane/water. Kinetic resolution of the racemic substrate was then attempted by employing catalysts generated from the [Ir(cod)Cl]2 precursor and single- and double-stranded DNA-PHOX conjugates. Good conversions were obtained in the presence of G-poor DNA/DNA and RNA/DNA hybrids bearing the PHOX moiety, indicating a potential role of the G-N7 site in the first coordination sphere. With all tested DNA-PHOX conjugates, the levels of enantioselectivity remained modest. The results described in this work provide useful information for understanding the influence of nucleic acid sequence and covalent tethering on the reaction outcome. These are the first reported applications of DNA-based ligands in organometallic catalysis and they build the fundamentals for further development of selective nucleic acid catalysts, by means of rational design and in vitro selection approaches

    In-vitro selection, characterization and application of a silver-specific DNAzyme

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    Evolution of novel DNA-based catalysts (DNAzymes) through in-vitro selection has expedited the research by manifolds, in both fundamental and analytical aspects of bio-nanotechnology. DNAzymes are attractive for their high stability, high catalytic efficiency and ease of modification. Among the known DNAzymes, those cleaving RNA have attracted most attention. Many of the known RNA-cleaving DNAzymes recruit multivalent metal ions for successful catalysis, while the catalytic involvement of monovalent metal ions yet remains underexplored. Before this thesis work, only a few monovalent ion-dependent DNAzymes are known (e.g. Na+-dependent EtNa and NaA43), and therefore, I was interested in exploring monovalent transition metals, such as silver. In this thesis, an Ag+-dependent selection experiment and its outcomes are described. In Chapter 1, relevant background information on DNA and DNAzymes was introduced, and the current state-of-the-art of the field was reviewed. In chapter 2, a new RNA-cleaving DNAzyme named Ag10c with a well-defined bulged hairpin structure was isolated after six rounds of in-vitro selection. The selection was performed using Ag+ as the intended target metal, and Na+ was present in the selection buffer to maintain the ionic strength of the buffer. This DNAzyme shows remarkable selectivity for Ag+, and attains a maximum speed of 0.41 min-1 in the presence of 10 µM Ag+ in buffer 50 mM MOPS (pH 7.5) and 200 mM NaNO3. This discovery expands the repertoire of metal-dependent RNA-cleaving DNAzymes and also draws much needed attention to the role of monovalent ions in DNAzyme catalysis. In chapter 3, the DNAzyme Ag10c was studied in extensive detail. The study revealed that most of the nucleotides in the catalytic loop are significant for activity. This study confirmed that Ag10c bears a well-defined silver aptamer in its catalytic loop, and it can fold into a compact structure by binding Ag+. Most nucleotides in the catalytic loop are highly conserved and mutations to them often dropped the activity by over 1000-fold. Using salt-dependent catalytic activity measurement, it was found that Ag10c was more active in buffers with higher NaCl concentration. However, other tested DNAzymes were inhibited by such salt. In addition, phosphorothioate modifications were made at the scissile phosphate. Based on these biochemical data, it was established that this Ag10c DNAzyme needs two metals for catalysis: one Na+ (or other group 1A metals or Mg2+) binds to the pro-Rp oxygen of the scissile phosphate, and two Ag+ ions bind cooperatively to Ag10c aptamer loop. It was also reported that Ag10c undergoes a single deprotonation step during catalysis. The investigation of Ag10c-Ag+ binding has been further characterized with fluorescence-based folding studies using 2-aminopurine as a probe. This study provides a new aptamer for Ag+ which is completely different from the well-known C-Ag+-C structure, and floats a new theme of DNAzyme catalysis that may be used by soft metals to escape interaction with the scissile phosphate and yet confer effective catalysis. In chapter 4, the DNAzyme Ag10c was used to develop a highly sensitive analytical probe for silver. This study reports a FRET-based system with the DNAzyme Ag10c for sensing low concentrations of Ag+ ions, with the limit of detection being 24.9 nM, which is far below the permissible limit of silver in water by WHO. The study exhibits that amongst the metals tested i.e. 100 mM, 10 mM and 100 μM of most of the group 1A metals, few of group 2A metals and many of the divalent transition metal ions respectively, the sensor is exceptionally selective for Ag+ ions. This study also demonstrates the robustness of the sensor in real world samples i.e. in Lake Huron water. This study puts forth a rare example of DNAzyme beacons being used for sensing of monovalent ions, and highlights the possibility of using DNAzyme beacons for sensing transition metal ions up to low nanomolar concentrations. In addition to the work pertaining to the silver DNAzyme, in-vitro selection was also performed on another toxic heavy metal, lead. A very short DNAzyme PbE22 was obtained. This DNAzyme consists of only 5 nucleotides in its catalytic loop and shows excellent selectivity for Pb2+. This part of information is presented in the Appendix A (chapter 6) of the thesis. Previously known Pb2+-specific DNAzymes, 17E and GR5, have also been characterized. By performing systematic mutations in their catalytic cores, and side-by-side comparison of both, four highly conserved nucleotides in both DNAzymes playing similar roles were identified and it was deciphered that they share the same activity pattern. This part of information is presented in the Appendix B (chapter 7) of the thesis. In summary, in-vitro selection is a powerful tool to obtain interesting metal-specific DNA sequences. This work has expanded the previous work to a monovalent heavy metal, silver. By studying Ag10c, a new mechanism of DNAzyme catalysis was revealed
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