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      Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids

      review-article
      Molecules
      MDPI
      modified nucleoside triphosphates, DNA polymerases, SELEX, DNAzymes, PEX, chemically modified nucleic acids

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          Abstract

          Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.

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          Most cited references127

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          Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.

          L Gold, C Tuerk (1990)
          High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
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            Functional nucleic acid sensors.

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              SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands.

              SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.
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                Author and article information

                Journal
                Molecules
                Molecules
                molecules
                Molecules
                MDPI
                1420-3049
                15 November 2012
                November 2012
                : 17
                : 11
                : 13569-13591
                Affiliations
                Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland; Email: hollenstein@ 123456dcb.unibe.ch ; Tel.: +41-316-314-372
                Article
                molecules-17-13569
                10.3390/molecules171113569
                6268876
                23154273
                88a860bd-3c62-4c94-8885-b01fee9188ce
                © 2012 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 21 September 2012
                : 07 November 2012
                : 09 November 2012
                Categories
                Review

                modified nucleoside triphosphates,dna polymerases,selex,dnazymes,pex,chemically modified nucleic acids

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