Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences
Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and in...
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Veröffentlicht in: | Genes & development 2015-04, Vol.29 (8), p.876-886 |
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creator | Wu, Bin Miskolci, Veronika Sato, Hanae Tutucci, Evelina Kenworthy, Charles A Donnelly, Sara K Yoon, Young J Cox, Dianne Singer, Robert H Hodgson, Louis |
description | Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts. |
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However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.</description><identifier>ISSN: 0890-9369</identifier><identifier>EISSN: 1549-5477</identifier><identifier>DOI: 10.1101/gad.259358.115</identifier><identifier>PMID: 25877922</identifier><language>eng</language><publisher>United States: Cold Spring Harbor Laboratory Press</publisher><subject>Animals ; Base Sequence - genetics ; Capsid Proteins - genetics ; Cell Line ; Cells, Cultured ; Codon - genetics ; Gene Expression ; Genetic Techniques ; Humans ; Levivirus - genetics ; Mice ; Nucleotide Motifs ; Repetitive Sequences, Nucleic Acid - genetics ; Resource/Methodology ; Saccharomyces cerevisiae - genetics</subject><ispartof>Genes & development, 2015-04, Vol.29 (8), p.876-886</ispartof><rights>2015 Wu et al.; Published by Cold Spring Harbor Laboratory Press.</rights><rights>2015</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c353t-c0de674e5e515036d5a130afecf963a7b2de8a4068f4f1e955fd0e380f5e2f3b3</citedby><cites>FETCH-LOGICAL-c353t-c0de674e5e515036d5a130afecf963a7b2de8a4068f4f1e955fd0e380f5e2f3b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403262/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403262/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25877922$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wu, Bin</creatorcontrib><creatorcontrib>Miskolci, Veronika</creatorcontrib><creatorcontrib>Sato, Hanae</creatorcontrib><creatorcontrib>Tutucci, Evelina</creatorcontrib><creatorcontrib>Kenworthy, Charles A</creatorcontrib><creatorcontrib>Donnelly, Sara K</creatorcontrib><creatorcontrib>Yoon, Young J</creatorcontrib><creatorcontrib>Cox, Dianne</creatorcontrib><creatorcontrib>Singer, Robert H</creatorcontrib><creatorcontrib>Hodgson, Louis</creatorcontrib><title>Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences</title><title>Genes & development</title><addtitle>Genes Dev</addtitle><description>Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.</description><subject>Animals</subject><subject>Base Sequence - genetics</subject><subject>Capsid Proteins - genetics</subject><subject>Cell Line</subject><subject>Cells, Cultured</subject><subject>Codon - genetics</subject><subject>Gene Expression</subject><subject>Genetic Techniques</subject><subject>Humans</subject><subject>Levivirus - genetics</subject><subject>Mice</subject><subject>Nucleotide Motifs</subject><subject>Repetitive Sequences, Nucleic Acid - genetics</subject><subject>Resource/Methodology</subject><subject>Saccharomyces cerevisiae - genetics</subject><issn>0890-9369</issn><issn>1549-5477</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkb1vFDEQxS1ERC6BlhK5pNnDH-uPbZBQRAJSJIpAbfns8Z3Rrr2svVHuv8fRhQg6qtFofu9pZh5CbynZUkroh731WyYGLnTrxQu0oaIfOtEr9RJtiB5IN3A5nKOLUn4SQiSR8hU6Z0IrNTC2QfnumHI6TnkteMo-huhsjTnhBco61oJjwoe4P3QhehhjPeI9JMDwMDegPII5NHaGGmu8BzwvuULT2ORxWt0IuTYhLvBrheSgvEZnwY4F3jzVS_Tj-vP3qy_d7bebr1efbjvHBa-dIx6k6kGAoIJw6YWlnNgALgySW7VjHrTtidShDxQGIYInwDUJAljgO36JPp5853U3gXeQ6mJHMy9xssvRZBvNv5MUD2af703fE84kawbvnwyW3HYv1UyxOBhHm6A9y1CplVSaEf4fqOoZYVLqhm5PqFtyKQuE540oMY-BmhaoOQXaetEE7_6-4xn_kyD_DVTEoG0</recordid><startdate>20150415</startdate><enddate>20150415</enddate><creator>Wu, Bin</creator><creator>Miskolci, Veronika</creator><creator>Sato, Hanae</creator><creator>Tutucci, Evelina</creator><creator>Kenworthy, Charles A</creator><creator>Donnelly, Sara K</creator><creator>Yoon, Young J</creator><creator>Cox, Dianne</creator><creator>Singer, Robert H</creator><creator>Hodgson, Louis</creator><general>Cold Spring Harbor Laboratory Press</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20150415</creationdate><title>Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences</title><author>Wu, Bin ; Miskolci, Veronika ; Sato, Hanae ; Tutucci, Evelina ; Kenworthy, Charles A ; Donnelly, Sara K ; Yoon, Young J ; Cox, Dianne ; Singer, Robert H ; Hodgson, Louis</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c353t-c0de674e5e515036d5a130afecf963a7b2de8a4068f4f1e955fd0e380f5e2f3b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Animals</topic><topic>Base Sequence - genetics</topic><topic>Capsid Proteins - genetics</topic><topic>Cell Line</topic><topic>Cells, Cultured</topic><topic>Codon - genetics</topic><topic>Gene Expression</topic><topic>Genetic Techniques</topic><topic>Humans</topic><topic>Levivirus - genetics</topic><topic>Mice</topic><topic>Nucleotide Motifs</topic><topic>Repetitive Sequences, Nucleic Acid - genetics</topic><topic>Resource/Methodology</topic><topic>Saccharomyces cerevisiae - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wu, Bin</creatorcontrib><creatorcontrib>Miskolci, Veronika</creatorcontrib><creatorcontrib>Sato, Hanae</creatorcontrib><creatorcontrib>Tutucci, Evelina</creatorcontrib><creatorcontrib>Kenworthy, Charles A</creatorcontrib><creatorcontrib>Donnelly, Sara K</creatorcontrib><creatorcontrib>Yoon, Young J</creatorcontrib><creatorcontrib>Cox, Dianne</creatorcontrib><creatorcontrib>Singer, Robert H</creatorcontrib><creatorcontrib>Hodgson, Louis</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Genes & development</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wu, Bin</au><au>Miskolci, Veronika</au><au>Sato, Hanae</au><au>Tutucci, Evelina</au><au>Kenworthy, Charles A</au><au>Donnelly, Sara K</au><au>Yoon, Young J</au><au>Cox, Dianne</au><au>Singer, Robert H</au><au>Hodgson, Louis</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences</atitle><jtitle>Genes & development</jtitle><addtitle>Genes Dev</addtitle><date>2015-04-15</date><risdate>2015</risdate><volume>29</volume><issue>8</issue><spage>876</spage><epage>886</epage><pages>876-886</pages><issn>0890-9369</issn><eissn>1549-5477</eissn><abstract>Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. 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subjects | Animals Base Sequence - genetics Capsid Proteins - genetics Cell Line Cells, Cultured Codon - genetics Gene Expression Genetic Techniques Humans Levivirus - genetics Mice Nucleotide Motifs Repetitive Sequences, Nucleic Acid - genetics Resource/Methodology Saccharomyces cerevisiae - genetics |
title | Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences |
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