Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing
Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of inter...
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| Veröffentlicht in: | Bioconjugate chemistry 2015-08, Vol.26 (8), p.1811-1817 |
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| container_title | Bioconjugate chemistry |
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| creator | Liu, Gary W Livesay, Brynn R Kacherovsky, Nataly A Cieslewicz, Maryelise Lutz, Emi Waalkes, Adam Jensen, Michael C Salipante, Stephen J Pun, Suzie H |
| description | Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective ( |
| doi_str_mv | 10.1021/acs.bioconjchem.5b00344 |
| format | Article |
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One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying “parasitic sequences” and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.</description><identifier>ISSN: 1043-1802</identifier><identifier>EISSN: 1520-4812</identifier><identifier>DOI: 10.1021/acs.bioconjchem.5b00344</identifier><identifier>PMID: 26161996</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Algorithms ; Animals ; Bacteriophages - genetics ; Binding sites ; Cells, Cultured ; Drug delivery systems ; Genetics ; High-Throughput Nucleotide Sequencing - methods ; Ligands ; Macrophages - cytology ; Macrophages - metabolism ; Mice ; Peptide Fragments - chemistry ; Peptide Fragments - genetics ; Peptide Fragments - metabolism ; Peptide Library ; Peptides</subject><ispartof>Bioconjugate chemistry, 2015-08, Vol.26 (8), p.1811-1817</ispartof><rights>Copyright © American Chemical Society</rights><rights>Copyright American Chemical Society Aug 19, 2015</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a489t-f561568b82261238da2781a2eb3aeca36e9c745ff0e686bdc15805f8133846643</citedby><cites>FETCH-LOGICAL-a489t-f561568b82261238da2781a2eb3aeca36e9c745ff0e686bdc15805f8133846643</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acs.bioconjchem.5b00344$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acs.bioconjchem.5b00344$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,776,780,881,2752,27053,27901,27902,56713,56763</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26161996$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Liu, Gary W</creatorcontrib><creatorcontrib>Livesay, Brynn R</creatorcontrib><creatorcontrib>Kacherovsky, Nataly A</creatorcontrib><creatorcontrib>Cieslewicz, Maryelise</creatorcontrib><creatorcontrib>Lutz, Emi</creatorcontrib><creatorcontrib>Waalkes, Adam</creatorcontrib><creatorcontrib>Jensen, Michael C</creatorcontrib><creatorcontrib>Salipante, Stephen J</creatorcontrib><creatorcontrib>Pun, Suzie H</creatorcontrib><title>Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing</title><title>Bioconjugate chemistry</title><addtitle>Bioconjugate Chem</addtitle><description>Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying “parasitic sequences” and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.</description><subject>Algorithms</subject><subject>Animals</subject><subject>Bacteriophages - genetics</subject><subject>Binding sites</subject><subject>Cells, Cultured</subject><subject>Drug delivery systems</subject><subject>Genetics</subject><subject>High-Throughput Nucleotide Sequencing - methods</subject><subject>Ligands</subject><subject>Macrophages - cytology</subject><subject>Macrophages - metabolism</subject><subject>Mice</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - metabolism</subject><subject>Peptide Library</subject><subject>Peptides</subject><issn>1043-1802</issn><issn>1520-4812</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU2P0zAQhi0EYpfCXwBLXLik-CuOc0FCy7Ks1MJKLGfLcSapq9QOdoIovx6XlmXhwsX22M-8M-MXoReULClh9LWxadm4YIPf2g3slmVDCBfiATqnJSOFUJQ9zGcieEEVYWfoSUpbQkhNFXuMzpikkta1PEc_LrvOWQd-wtdtXl2OzOSCx6HD6zk6D3jN8NrYGMaN6QHfwDi5FvCtiT1Mzvd45Xrj24SbPb75hbxzaRzMHudb_BG-T8UVeIhH2c_wdQZvc95T9KgzQ4Jnp32Bvry_vL34UKw-XV1fvF0VRqh6KrpS0lKqRrHcNeOqNaxS1DBouAFruITaVqLsOgJSyaa1tFSk7BTlXAkpBV-gN0fdcW520No8ZTSDHqPbmbjXwTj994t3G92Hb1pIQZSqs8Crk0AMufk06Z1LFobBeAhz0rQiZcW5zBUX6OU_6DbM0efxDpSsCOOVylR1pPKnphShu2uGEn3wV2d_9T1_9cnfnPn8_ix3eb8NzQA_AgeFP7X_I_sTb5y4jA</recordid><startdate>20150819</startdate><enddate>20150819</enddate><creator>Liu, Gary W</creator><creator>Livesay, Brynn R</creator><creator>Kacherovsky, Nataly A</creator><creator>Cieslewicz, Maryelise</creator><creator>Lutz, Emi</creator><creator>Waalkes, Adam</creator><creator>Jensen, Michael C</creator><creator>Salipante, Stephen J</creator><creator>Pun, Suzie H</creator><general>American Chemical Society</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>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20150819</creationdate><title>Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing</title><author>Liu, Gary W ; Livesay, Brynn R ; Kacherovsky, Nataly A ; Cieslewicz, Maryelise ; Lutz, Emi ; Waalkes, Adam ; Jensen, Michael C ; Salipante, Stephen J ; Pun, Suzie H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a489t-f561568b82261238da2781a2eb3aeca36e9c745ff0e686bdc15805f8133846643</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Algorithms</topic><topic>Animals</topic><topic>Bacteriophages - genetics</topic><topic>Binding sites</topic><topic>Cells, Cultured</topic><topic>Drug delivery systems</topic><topic>Genetics</topic><topic>High-Throughput Nucleotide Sequencing - methods</topic><topic>Ligands</topic><topic>Macrophages - cytology</topic><topic>Macrophages - metabolism</topic><topic>Mice</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - metabolism</topic><topic>Peptide Library</topic><topic>Peptides</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Liu, Gary W</creatorcontrib><creatorcontrib>Livesay, Brynn R</creatorcontrib><creatorcontrib>Kacherovsky, Nataly A</creatorcontrib><creatorcontrib>Cieslewicz, Maryelise</creatorcontrib><creatorcontrib>Lutz, Emi</creatorcontrib><creatorcontrib>Waalkes, Adam</creatorcontrib><creatorcontrib>Jensen, Michael C</creatorcontrib><creatorcontrib>Salipante, Stephen J</creatorcontrib><creatorcontrib>Pun, Suzie H</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Bioconjugate chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Liu, Gary W</au><au>Livesay, Brynn R</au><au>Kacherovsky, Nataly A</au><au>Cieslewicz, Maryelise</au><au>Lutz, Emi</au><au>Waalkes, Adam</au><au>Jensen, Michael C</au><au>Salipante, Stephen J</au><au>Pun, Suzie H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing</atitle><jtitle>Bioconjugate chemistry</jtitle><addtitle>Bioconjugate Chem</addtitle><date>2015-08-19</date><risdate>2015</risdate><volume>26</volume><issue>8</issue><spage>1811</spage><epage>1817</epage><pages>1811-1817</pages><issn>1043-1802</issn><eissn>1520-4812</eissn><abstract>Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying “parasitic sequences” and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. 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| ispartof | Bioconjugate chemistry, 2015-08, Vol.26 (8), p.1811-1817 |
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| source | MEDLINE; American Chemical Society Journals |
| subjects | Algorithms Animals Bacteriophages - genetics Binding sites Cells, Cultured Drug delivery systems Genetics High-Throughput Nucleotide Sequencing - methods Ligands Macrophages - cytology Macrophages - metabolism Mice Peptide Fragments - chemistry Peptide Fragments - genetics Peptide Fragments - metabolism Peptide Library Peptides |
| title | Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing |
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