Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors
The epigenetic DNA nucleobases 5-methylcytosine (5mC) and 4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for dist...
Gespeichert in:
Veröffentlicht in: | Philosophical transactions of the Royal Society of London. Series B. Biological sciences 2018-06, Vol.373 (1748), p.20170078 |
---|---|
Hauptverfasser: | , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
container_end_page | |
---|---|
container_issue | 1748 |
container_start_page | 20170078 |
container_title | Philosophical transactions of the Royal Society of London. Series B. Biological sciences |
container_volume | 373 |
creator | Rathi, Preeti Maurer, Sara Summerer, Daniel |
description | The epigenetic DNA nucleobases 5-methylcytosine (5mC) and
4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. |
doi_str_mv | 10.1098/rstb.2017.0078 |
format | Article |
fullrecord | <record><control><sourceid>pubmed_cross</sourceid><recordid>TN_cdi_crossref_primary_10_1098_rstb_2017_0078</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>29685980</sourcerecordid><originalsourceid>FETCH-LOGICAL-c1478-7e33803349b54b1076a99c50d3117429c2ef13cd3cfa44c76e252bc0aece89ff3</originalsourceid><addsrcrecordid>eNo9kMtOwzAQAC0EoqVw5Yj8AwnrR2L7WPGWqnIAzpHjrIuhTSo7IOXvSVTgtNJqZ7QaQi4Z5AyMvo6pr3MOTOUASh-ROZOKZdwoOCZzMCXPtBTljJyl9AEAplDylMy4KXVhNMzJ5gW36PrwjTSi6zZt6EPX0s7TNZXZDvv3YeuGvkuhRRpaerte0nqg2G7GBUZsaB9tm1wM-wnM7OSyfRezbfhEit6P9i6mc3Li7Tbhxe9ckLf7u9ebx2z1_PB0s1xlbnxcZwqF0CCENHUhawaqtMa4AhrBmJLcOI6eCdcI562UTpXIC147sOhQG-_FguQHr4tdShF9tY9hZ-NQMaimYtVUrJqKVVOxEbg6APuveofN__lfIvEDLe5pSA</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors</title><source>Jstor Complete Legacy</source><source>PubMed Central Free</source><source>MEDLINE</source><creator>Rathi, Preeti ; Maurer, Sara ; Summerer, Daniel</creator><creatorcontrib>Rathi, Preeti ; Maurer, Sara ; Summerer, Daniel</creatorcontrib><description>The epigenetic DNA nucleobases 5-methylcytosine (5mC) and
4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.</description><identifier>ISSN: 0962-8436</identifier><identifier>EISSN: 1471-2970</identifier><identifier>DOI: 10.1098/rstb.2017.0078</identifier><identifier>PMID: 29685980</identifier><language>eng</language><publisher>England</publisher><subject>5-Methylcytosine - chemistry ; DNA - chemistry ; Genetic Engineering ; Transcription Activator-Like Effectors - chemistry</subject><ispartof>Philosophical transactions of the Royal Society of London. Series B. Biological sciences, 2018-06, Vol.373 (1748), p.20170078</ispartof><rights>2018 The Author(s).</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c1478-7e33803349b54b1076a99c50d3117429c2ef13cd3cfa44c76e252bc0aece89ff3</citedby><cites>FETCH-LOGICAL-c1478-7e33803349b54b1076a99c50d3117429c2ef13cd3cfa44c76e252bc0aece89ff3</cites><orcidid>0000-0002-3019-7241</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29685980$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Rathi, Preeti</creatorcontrib><creatorcontrib>Maurer, Sara</creatorcontrib><creatorcontrib>Summerer, Daniel</creatorcontrib><title>Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors</title><title>Philosophical transactions of the Royal Society of London. Series B. Biological sciences</title><addtitle>Philos Trans R Soc Lond B Biol Sci</addtitle><description>The epigenetic DNA nucleobases 5-methylcytosine (5mC) and
4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.</description><subject>5-Methylcytosine - chemistry</subject><subject>DNA - chemistry</subject><subject>Genetic Engineering</subject><subject>Transcription Activator-Like Effectors - chemistry</subject><issn>0962-8436</issn><issn>1471-2970</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kMtOwzAQAC0EoqVw5Yj8AwnrR2L7WPGWqnIAzpHjrIuhTSo7IOXvSVTgtNJqZ7QaQi4Z5AyMvo6pr3MOTOUASh-ROZOKZdwoOCZzMCXPtBTljJyl9AEAplDylMy4KXVhNMzJ5gW36PrwjTSi6zZt6EPX0s7TNZXZDvv3YeuGvkuhRRpaerte0nqg2G7GBUZsaB9tm1wM-wnM7OSyfRezbfhEit6P9i6mc3Li7Tbhxe9ckLf7u9ebx2z1_PB0s1xlbnxcZwqF0CCENHUhawaqtMa4AhrBmJLcOI6eCdcI562UTpXIC147sOhQG-_FguQHr4tdShF9tY9hZ-NQMaimYtVUrJqKVVOxEbg6APuveofN__lfIvEDLe5pSA</recordid><startdate>20180605</startdate><enddate>20180605</enddate><creator>Rathi, Preeti</creator><creator>Maurer, Sara</creator><creator>Summerer, Daniel</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><orcidid>https://orcid.org/0000-0002-3019-7241</orcidid></search><sort><creationdate>20180605</creationdate><title>Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors</title><author>Rathi, Preeti ; Maurer, Sara ; Summerer, Daniel</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c1478-7e33803349b54b1076a99c50d3117429c2ef13cd3cfa44c76e252bc0aece89ff3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>5-Methylcytosine - chemistry</topic><topic>DNA - chemistry</topic><topic>Genetic Engineering</topic><topic>Transcription Activator-Like Effectors - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rathi, Preeti</creatorcontrib><creatorcontrib>Maurer, Sara</creatorcontrib><creatorcontrib>Summerer, Daniel</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>Philosophical transactions of the Royal Society of London. Series B. Biological sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rathi, Preeti</au><au>Maurer, Sara</au><au>Summerer, Daniel</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors</atitle><jtitle>Philosophical transactions of the Royal Society of London. Series B. Biological sciences</jtitle><addtitle>Philos Trans R Soc Lond B Biol Sci</addtitle><date>2018-06-05</date><risdate>2018</risdate><volume>373</volume><issue>1748</issue><spage>20170078</spage><pages>20170078-</pages><issn>0962-8436</issn><eissn>1471-2970</eissn><abstract>The epigenetic DNA nucleobases 5-methylcytosine (5mC) and
4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.</abstract><cop>England</cop><pmid>29685980</pmid><doi>10.1098/rstb.2017.0078</doi><orcidid>https://orcid.org/0000-0002-3019-7241</orcidid><oa>free_for_read</oa></addata></record> |
fulltext | fulltext |
identifier | ISSN: 0962-8436 |
ispartof | Philosophical transactions of the Royal Society of London. Series B. Biological sciences, 2018-06, Vol.373 (1748), p.20170078 |
issn | 0962-8436 1471-2970 |
language | eng |
recordid | cdi_crossref_primary_10_1098_rstb_2017_0078 |
source | Jstor Complete Legacy; PubMed Central Free; MEDLINE |
subjects | 5-Methylcytosine - chemistry DNA - chemistry Genetic Engineering Transcription Activator-Like Effectors - chemistry |
title | Selective recognition of N 4-methylcytosine in DNA by engineered transcription-activator-like effectors |
url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T11%3A47%3A39IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-pubmed_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Selective%20recognition%20of%20N%204-methylcytosine%20in%20DNA%20by%20engineered%20transcription-activator-like%20effectors&rft.jtitle=Philosophical%20transactions%20of%20the%20Royal%20Society%20of%20London.%20Series%20B.%20Biological%20sciences&rft.au=Rathi,%20Preeti&rft.date=2018-06-05&rft.volume=373&rft.issue=1748&rft.spage=20170078&rft.pages=20170078-&rft.issn=0962-8436&rft.eissn=1471-2970&rft_id=info:doi/10.1098/rstb.2017.0078&rft_dat=%3Cpubmed_cross%3E29685980%3C/pubmed_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_id=info:pmid/29685980&rfr_iscdi=true |