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      Transcription of Click-Linked DNA in Human Cells**

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          Abstract

          Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase-based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click-linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error-free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER-deficient human cell line. This is the first example of a non-natural DNA linker being functional in a eukaryotic cell.

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          Most cited references 38

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          Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.

          Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
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            Cancer epigenetics: from mechanism to therapy.

            The epigenetic regulation of DNA-templated processes has been intensely studied over the last 15 years. DNA methylation, histone modification, nucleosome remodeling, and RNA-mediated targeting regulate many biological processes that are fundamental to the genesis of cancer. Here, we present the basic principles behind these epigenetic pathways and highlight the evidence suggesting that their misregulation can culminate in cancer. This information, along with the promising clinical and preclinical results seen with epigenetic drugs against chromatin regulators, signifies that it is time to embrace the central role of epigenetics in cancer. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Epigenetic modifications and human disease.

              Epigenetics is one of the most rapidly expanding fields in biology. The recent characterization of a human DNA methylome at single nucleotide resolution, the discovery of the CpG island shores, the finding of new histone variants and modifications, and the unveiling of genome-wide nucleosome positioning maps highlight the accelerating speed of discovery over the past two years. Increasing interest in epigenetics has been accompanied by technological breakthroughs that now make it possible to undertake large-scale epigenomic studies. These allow the mapping of epigenetic marks, such as DNA methylation, histone modifications and nucleosome positioning, which are critical for regulating gene and noncoding RNA expression. In turn, we are learning how aberrant placement of these epigenetic marks and mutations in the epigenetic machinery is involved in disease. Thus, a comprehensive understanding of epigenetic mechanisms, their interactions and alterations in health and disease, has become a priority in biomedical research.
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                Author and article information

                Journal
                Angew Chem Int Ed Engl
                Angew. Chem. Int. Ed. Engl
                anie
                Angewandte Chemie (International Ed. in English)
                Wiley-VCH Verlag GmbH & Co (KGaA, Weinheim )
                1433-7851
                1521-3773
                24 February 2014
                22 January 2014
                : 53
                : 9
                : 2362-2365
                Affiliations
                Chemistry, University of Southampton Southampton, SO17 1BJ (UK)
                Cancer Sciences, Faculty of Medicine, University of Southampton Southampton, SO16 6YD (UK)
                Dept. of Science and Mathematics, Suez University Suez, 43721 (Egypt)
                Department of Chemistry, University of Oxford, Chemistry Research Laboratory Oxford, OX1 3TA (UK)
                Author notes
                [*]Dr. A. Tavassoli Chemistry, University of Southampton Southampton, SO17 1BJ (UK) E-mail: a.tavassoli@ 123456soton.ac.uk
                [[+]]

                These authors contributed equally to this work.

                [[**]]

                This work was supported by Cancer Research UK (Career Establishment Award 10263 to A.T.); Breast Cancer Campaign (grant 2010NovPR12 to J.P.B. and A.T.), and the BBSRC (sLOLA grant BB/J001694/1 to T.B. and A.T.). We thank Prof. Roger Tsien for providing the mCherry gene and Dr. Josephine Corsi for discussions that led to initiation of this work.

                Article
                10.1002/anie.201308691
                4016740
                24452865
                © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Categories
                Synthetic Biology

                Chemistry

                click chemistry, dna ligation, gene technology, nucleic acids, synthetic biology

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