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      Catalytic Nucleic Acids: Biochemistry, Chemical Biology, Biosensors, and Nanotechnology

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      1 , 1 ,
      iScience
      Elsevier
      Chemistry, Biochemistry, Biochemistry Applications, Enzymology, Biomolecules, Nanotechnology

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          Abstract

          Since the initial discovery of ribozymes in the early 1980s, catalytic nucleic acids have been used in different areas. Compared with protein enzymes, catalytic nucleic acids are programmable in structure, easy to modify, and more stable especially for DNA. We take a historic view to summarize a few main interdisciplinary areas of research on nucleic acid enzymes that may have broader impacts. Early efforts on ribozymes in the 1980s have broken the notion that all enzymes are proteins, supplying new evidence for the RNA world hypothesis. In 1994, the first catalytic DNA (DNAzyme) was reported. Since 2000, the biosensor applications of DNAzymes have emerged and DNAzymes are particularly useful for detecting metal ions, a challenging task for enzymes and antibodies. Combined with nanotechnology, DNAzymes are key building elements for switches allowing dynamic control of materials assembly. The search for new DNAzymes and ribozymes is facilitated by developments in DNA sequencing and computational algorithms, further broadening our fundamental understanding of their biochemistry.

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          Abstract

          Chemistry; Biochemistry; Biochemistry Applications; Enzymology; Biomolecules; Nanotechnology

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          Functional nucleic acid sensors.

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            Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena.

            In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.
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              A DNA enzyme that cleaves RNA.

              Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.
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                Author and article information

                Contributors
                Journal
                iScience
                iScience
                iScience
                Elsevier
                2589-0042
                02 January 2020
                24 January 2020
                02 January 2020
                : 23
                : 1
                : 100815
                Affiliations
                [1 ]Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON N2L 3G1, Canada
                Author notes
                []Corresponding author liujw@ 123456uwaterloo.ca
                Article
                S2589-0042(19)30561-9 100815
                10.1016/j.isci.2019.100815
                6962706
                31954323
                5c997036-4cbf-4bda-8653-8b06c583fad6
                © 2019 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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                chemistry,biochemistry,biochemistry applications,enzymology,biomolecules,nanotechnology

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