{"id":153,"date":"2021-05-18T01:54:59","date_gmt":"2021-05-18T01:54:59","guid":{"rendered":"https:\/\/chem.sites.mtu.edu\/amidities\/index.php\/academics\/"},"modified":"2023-11-15T19:12:13","modified_gmt":"2023-11-15T19:12:13","slug":"protocol","status":"publish","type":"page","link":"https:\/\/chem.sites.mtu.edu\/amidities\/protocol\/","title":{"rendered":"Protocol"},"content":{"rendered":"\t\t
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Long ODN synthesis and CBP purification<\/h1>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t
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PRINCIPLE<\/h6>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\n\t\t\t\n\t\t\t\t\t\t<\/span>\n\t\t<\/div>\n\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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During oligodeoxynucleotide (ODN) synthesis, the failure sequences are capped with Ac2<\/sub>O and full-length sequences are tagged with a polymerizable nucleoside (PdN<\/strong>) phosphoramidite (Figure). After cleavage and deprotection, the crude contains the full-length sequences (the desired product) with a polymerizable group, failure sequences and small molecules. For purification, the full-length sequences are co-polymerized into a polyacrylamide gel. Failure sequences and small molecules are washed away. The product is cleaved and extracted, and precipitated with n<\/em>BuOH.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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MATERIALS (for purification of 1 \u03bcmol or less ODN)<\/h6>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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Polymerizable dA amidite (PdA<\/strong>, MW 1156.3, 29 mg, 25 \u03bcmol; for sequences with a dA at 5\u2032-end)<\/p>

Polymerizable dC amidite (PdC<\/strong>, MW 1070.2, 27 mg, 25 \u03bcmol; for sequences with a dC at 5\u2032-end)<\/p>

Polymerizable dG amidite (PdG<\/strong>, MW 1138.3, 28 mg, 25 \u03bcmol; for sequences with a dG at 5\u2032-end)<\/p>

Polymerizable dT amidite (PdT<\/strong>, MW 1043.2, 26 mg, 25 \u03bcmol; for sequences with a dT at 5\u2032-end)<\/p>

\u00a0<\/p>

Polymerization mix [PM<\/strong>, N<\/em>,N<\/em>-dimethylacrylamide, MW 99.1, d 0.96, 327 mg, 340 \u03bcl, 3.3 mmol; N<\/em>,N’<\/em>-methylenebis(acrylamide), MW 154.2, 17 mg, 110 \u03bcmol; polyacrylamide cross-linking ratio 20:1; sodium acrylate, MW 94.0, 3 mg, 33 \u00b5mol]<\/p>

Polymerization initiator A (PIA<\/strong>, ammonium persulfate, MW 228.2, 10 mg, 44 \u03bcmol)<\/p>

Polymerization initiator B (PIB<\/strong>, N,N,N’,N’<\/em>-tetramethylethylenediamine, MW 116.2, d 0.78, 15.5 mg, 20 \u03bcl, 132 \u03bcmol)<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t\t\t

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PROTOCOL (more details in reference 1)<\/h6>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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Synthesize ODN using PdN to tag full-length sequences<\/strong> <\/em><\/p>

  1. Add 250 \u03bcl dry acetonitrile to PdN<\/strong> (0.1 M). Shake gently to dissolve.<\/li>
  2. Conduct ODN synthesis on a synthesizer using Ac2<\/sub>O or Pac2<\/sub>O for capping.<\/li>
  3. Incorporate the last nucleotide using PdN<\/strong> Coupling time should be 5-10 min. No deblocking in this cycle.<\/li>
  4. Perform cleavage and deprotection as usual (UltraMild: conc. NH4<\/sub>OH, rt, 2 h; normal conditions: conc. NH4<\/sub>OH, 55 \u00b0C, 10-15 h; or conc. NH4<\/sub>OH\/MeNH2<\/sub> 1\/1, rt, ~3 h).<\/li>
  5. Transfer ODN solution into a 1.5 ml centrifuge tube. Add ~0.1 mg K2<\/sub>CO3<\/sub>, and evaporate volatiles. The residue is the crude ODN with full-length sequences being tagged with PdN<\/strong>.<\/li><\/ol>

    Polymerize full-length sequences<\/strong> <\/em><\/p>

    1. Add 170 \u03bcl ddH2<\/sub>O to PM<\/strong>. Vortex and spin briefly.<\/li>
    2. Add 190 \u03bcl ddH2<\/sub>O to PIA<\/strong>. Vortex and spin briefly.<\/li>
    3. Add 190 \u03bcl ddH2<\/sub>O to PIB<\/strong>. Vortex and spin briefly.<\/li>
    4. Add 50 \u03bcl ddH2<\/sub>O to crude ODN (or a portion of the crude). Vortex and spin briefly.<\/li>
    5. Add 12 \u03bcl PM<\/strong> Vortex and spin briefly.<\/li>
    6. Add PIA<\/strong> and PIB<\/strong> (5 \u03bcl), vortex (~1 sec) and spin (~5 sec) immediately.<\/li>
    7. Cap the tube and let stand at rt for 1 h.<\/li><\/ol>

      Wash away failure sequences and other impurities<\/em><\/strong><\/p>

      1. Transfer the gel to a 50 ml centrifuge tube and cut it into pieces (the pieces should be large enough for transfer).<\/li>
      2. Add ~30 ml 20% NaOAc, shake gently at rt overnight.<\/li>
      3. Remove supernatant.<\/li>
      4. Add ~30 ml 5% Et3<\/sub>N, shake gently at rt overnight.<\/li>
      5. Remove supernatant.<\/li>
      6. Transfer the gel pieces onto the filter in a 2 ml centrifugal filter unit.<\/li>
      7. Wash the gel with ddH2<\/sub>O (0.5 ml \u00d7 5).<\/li><\/ol>

        \u00a0<\/em>Cleave and extract full-length sequences<\/em><\/strong><\/p>

        1. Add minimum 80% AcOH to cover the gel (~200 \u03bcl), wait for 3-5 min, spin.<\/li>
        2. Dilute the filtrate with ddH2<\/sub>O (~800 \u00b5l) immediately.<\/li>
        3. Repeat acid cleavage two times.<\/li>
        4. Wash the gel with ddH2<\/sub>O (~250 \u00b5l \u00d7 3).<\/li>
        5. Stop the bottom of the filtering unit with parafilm.<\/li>
        6. Add ddH2<\/sub>O (~500 \u00b5l) to the gel, and gently shake the filtering unit at rt overnight.<\/li>
        7. Combine the cleavage and washing filtrates.<\/li>
        8. Evaporate to dryness. This gives the purified ODN. The following prescription step is optional (if precipitation is omitted, add ddH2<\/sub>O to the ODN and evaporate again to ensure residue acid is removed).<\/li><\/ol>

          Precipitate (optional)<\/em><\/strong><\/p>

          1. Add 50 \u00b5l conc. NH4<\/sub> Cap, vortex and spin.<\/li>
          2. Add 450 \u03bcl n<\/em> Close the cap.<\/li>
          3. Vortex the tube for ~30 sec and then centrifuge at ~14.5k rpm (14.1k \u00d7 g) for ~5 min.<\/li>
          4. Remove the supernatant with a pipette, evaporate residue solvent. This gives the purified ODN.<\/li><\/ol>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t<\/section>\n\t\t
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            REFERENCES AND NOTES<\/em><\/strong><\/p>

            1. ChemRxiv [preprint]<\/em> 2023 <\/strong>doi:10.26434\/chemrxiv-2023-79qj3<\/pre>
            2. Org Process Res Dev<\/em> 2018 <\/strong>22 <\/em>1282-1288 doi:10.1021\/acs.oprd.8b00209<\/pre>
            3. Green Chem<\/em> 2016 <\/strong>18 <\/em>1125-1136 doi:10.1039\/c5gc01762a<\/pre>
            4. Org Lett<\/em> 2014 <\/strong>16 <\/em>1290-1293 doi:10.1021\/ol403426u<\/pre>
            The steps for washing away failure sequences may not need to be as extensive as described. Smaller washing volume and shorter washing time may be sufficient, and it may be carried out in a centrifugal filtering unit.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t","protected":false},"excerpt":{"rendered":"
            Long ODN synthesis and CBP purification PRINCIPLE During oligodeoxynucleotide (ODN) synthesis, the failure sequences are capped with Ac2O and full-length sequences are tagged with a polymerizable nucleoside (PdN) phosphoramidite (Figure). After cleavage and deprotection, the crude contains the full-length sequences (the desired product) with a polymerizable group, failure sequences and small molecules. For purification, the […]<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"site-sidebar-layout":"no-sidebar","site-content-layout":"page-builder","ast-site-content-layout":"default","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"disabled","ast-breadcrumbs-content":"disabled","ast-featured-img":"disabled","footer-sml-layout":"","ast-disable-related-posts":"","theme-transparent-header-meta":"default","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"footnotes":""},"class_list":["post-153","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/pages\/153","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/comments?post=153"}],"version-history":[{"count":8,"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/pages\/153\/revisions"}],"predecessor-version":[{"id":706,"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/pages\/153\/revisions\/706"}],"wp:attachment":[{"href":"https:\/\/chem.sites.mtu.edu\/amidities\/wp-json\/wp\/v2\/media?parent=153"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}