Photocleavable Biotin Phosphoramidite for 5′-End-Labeling, Affinity Purification and Phosphorylation of Synthetic Oligonucleotides
We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5′-terminal phos...
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| Veröffentlicht in: | Nucleic acids research 1996-01, Vol.24 (2), p.361-366 |
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| container_title | Nucleic acids research |
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| creator | Olejnik, Jerzy Krzymanska-Olejnik, Edyta Rothschild, Kenneth J. |
| description | We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5′-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5′-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300–350 nm light the 5′-PCB moiety is cleaved with high efficiency in |
| doi_str_mv | 10.1093/nar/24.2.361 |
| format | Article |
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This reagent introduces a photocleavable biotin label (PCB) on the 5′-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5′-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300–350 nm light the 5′-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5′-phosphorylated oligonucleotides into solution. 5′-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50- and 60mer oligonucleotides.</description><identifier>ISSN: 0305-1048</identifier><identifier>ISSN: 1362-4962</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/24.2.361</identifier><identifier>PMID: 8628663</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Biotin - analogs & derivatives ; Biotin - chemical synthesis ; Biotin - chemistry ; Oligonucleotides - chemical synthesis ; Oligonucleotides - chemistry ; Oligonucleotides - isolation & purification ; Oligonucleotides - metabolism ; Organophosphorus Compounds - chemical synthesis ; Organophosphorus Compounds - chemistry ; Phosphorylation ; Photolysis ; Ultraviolet Rays</subject><ispartof>Nucleic acids research, 1996-01, Vol.24 (2), p.361-366</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c544t-b23b2422723a3a01afd99f82fcc336bf5b1f78c8c72b4f9a0084ebebd93361533</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC145639/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC145639/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27923,27924,53790,53792</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8628663$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Olejnik, Jerzy</creatorcontrib><creatorcontrib>Krzymanska-Olejnik, Edyta</creatorcontrib><creatorcontrib>Rothschild, Kenneth J.</creatorcontrib><title>Photocleavable Biotin Phosphoramidite for 5′-End-Labeling, Affinity Purification and Phosphorylation of Synthetic Oligonucleotides</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Research</addtitle><description>We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5′-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5′-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300–350 nm light the 5′-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5′-phosphorylated oligonucleotides into solution. 5′-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50- and 60mer oligonucleotides.</description><subject>Biotin - analogs & derivatives</subject><subject>Biotin - chemical synthesis</subject><subject>Biotin - chemistry</subject><subject>Oligonucleotides - chemical synthesis</subject><subject>Oligonucleotides - chemistry</subject><subject>Oligonucleotides - isolation & purification</subject><subject>Oligonucleotides - metabolism</subject><subject>Organophosphorus Compounds - chemical synthesis</subject><subject>Organophosphorus Compounds - chemistry</subject><subject>Phosphorylation</subject><subject>Photolysis</subject><subject>Ultraviolet Rays</subject><issn>0305-1048</issn><issn>1362-4962</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc9uEzEQxi1EVdLCjSvSnjh1U_9b7_rAoVQtQY1oJUBEXCzbaycGxw62tyI3DjwRj8STsFWiCE6cRprfN9_M6APgOYJTBDk5DzKdYzrFU8LQIzBBhOGacoYfgwkksKkRpN0TcJLzFwgRRQ09Bscdwx1jZAJ-3q1iidobeS-VN9VrF4sL1djNm1VMcu16V0xlY6qa3z9-1Vehr-dSGe_C8qy6sNYFV7bV3ZCcdVoWF0MlQ38w2PpdL9rq_TaUlSlOV7feLWMYxq3jst7kp-DISp_Ns309BR-vrz5czur57Zu3lxfzWjeUllphojDFuMVEEgmRtD3ntsNWa0KYso1Ctu10p1usqOUSwo4aZVTPR4waQk7Bq53vZlBr02sTSpJebJJby7QVUTrxLwluJZbxXiDaMMLH-Zf7-RS_DSYXsXZZG-9lMHHIAnHC2qZl_xe2EHHIHxzPdkKdYs7J2MMxCIqHdMWYrsBUYDG-MMpf_P3AQbyPc-T1jrtczPcDlumrYC1pGzFbfBbvbuiCf5rNxYL8ATt_tRg</recordid><startdate>19960115</startdate><enddate>19960115</enddate><creator>Olejnik, Jerzy</creator><creator>Krzymanska-Olejnik, Edyta</creator><creator>Rothschild, Kenneth J.</creator><general>Oxford University Press</general><scope>BSCLL</scope><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>7TM</scope><scope>5PM</scope></search><sort><creationdate>19960115</creationdate><title>Photocleavable Biotin Phosphoramidite for 5′-End-Labeling, Affinity Purification and Phosphorylation of Synthetic Oligonucleotides</title><author>Olejnik, Jerzy ; Krzymanska-Olejnik, Edyta ; Rothschild, Kenneth J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c544t-b23b2422723a3a01afd99f82fcc336bf5b1f78c8c72b4f9a0084ebebd93361533</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>Biotin - analogs & derivatives</topic><topic>Biotin - chemical synthesis</topic><topic>Biotin - chemistry</topic><topic>Oligonucleotides - chemical synthesis</topic><topic>Oligonucleotides - chemistry</topic><topic>Oligonucleotides - isolation & purification</topic><topic>Oligonucleotides - metabolism</topic><topic>Organophosphorus Compounds - chemical synthesis</topic><topic>Organophosphorus Compounds - chemistry</topic><topic>Phosphorylation</topic><topic>Photolysis</topic><topic>Ultraviolet Rays</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Olejnik, Jerzy</creatorcontrib><creatorcontrib>Krzymanska-Olejnik, Edyta</creatorcontrib><creatorcontrib>Rothschild, Kenneth J.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Olejnik, Jerzy</au><au>Krzymanska-Olejnik, Edyta</au><au>Rothschild, Kenneth J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Photocleavable Biotin Phosphoramidite for 5′-End-Labeling, Affinity Purification and Phosphorylation of Synthetic Oligonucleotides</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Research</addtitle><date>1996-01-15</date><risdate>1996</risdate><volume>24</volume><issue>2</issue><spage>361</spage><epage>366</epage><pages>361-366</pages><issn>0305-1048</issn><issn>1362-4962</issn><eissn>1362-4962</eissn><abstract>We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5′-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5′-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300–350 nm light the 5′-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5′-phosphorylated oligonucleotides into solution. 5′-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50- and 60mer oligonucleotides.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>8628663</pmid><doi>10.1093/nar/24.2.361</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0305-1048 |
| ispartof | Nucleic acids research, 1996-01, Vol.24 (2), p.361-366 |
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| source | MEDLINE; Oxford Journals Open Access Collection; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Biotin - analogs & derivatives Biotin - chemical synthesis Biotin - chemistry Oligonucleotides - chemical synthesis Oligonucleotides - chemistry Oligonucleotides - isolation & purification Oligonucleotides - metabolism Organophosphorus Compounds - chemical synthesis Organophosphorus Compounds - chemistry Phosphorylation Photolysis Ultraviolet Rays |
| title | Photocleavable Biotin Phosphoramidite for 5′-End-Labeling, Affinity Purification and Phosphorylation of Synthetic Oligonucleotides |
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