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POST-SYNTHETIC MODIFICATION OF NUCLEIC ACIDS BY INVERSE DIELS-ALDER REACTION

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  • Publication Date:
    April 4, 2013
  • Additional Information
    • Document Number:
      20130085271
    • Appl. No:
      13/557698
    • Application Filed:
      July 25, 2012
    • Abstract:
      The present invention concerns a method and a kit for the post-synthetic modification of nucleic acids via an inverse Diels-Alder reaction.
    • Claim:
      1. A method for the post-synthetic modification of nucleic acids, comprising the following steps: (a) preparing norbornene or trans-cyclooctene-modified oligonucleotides (b) preparing modified tetrazines, and (c) reacting the norbornene-modified oligonucleotides with the modified tetrazines via inverse Diels Alder reaction.
    • Claim:
      2. The method of claim 1, wherein the tetrazines are modified with a compound selected from the group consisting of fluorescent, luminescent or phosphorescent dyes, and affinity tags.
    • Claim:
      3. The method of claim 1, wherein the oligonucleotide is singly or multiply modified.
    • Claim:
      4. The method of claim 3, wherein the oligonucleotide is modified terminally and/or internally.
    • Claim:
      5. The method of claim 4, wherein the terminal modification is 3′and/or 5′.
    • Claim:
      6. The method of claim 1, wherein the oligonucleotide has a length between 3 and 500 nucleotides.
    • Claim:
      7. The method of claim 1, wherein the oligonucleotide is single-stranded or double-stranded DNA or RNA, a nucleic acid analog or chimera thereof with DNA and/or RNA or an enyzmatically modified PCR product.
    • Claim:
      8. The method of claim 1, wherein the norbornene- or trans-cyclooctene-modified oligonucleotide is prepared by reacting an oligonucleotide with one or more of the following compounds: [chemical expression included] [chemical expression included]
    • Claim:
      9. The method of claim 1, wherein the tetrazine is labelled with one or more of fluorescent, luminescent or phosphorescent dyes, or tagged with one or more affinity tags.
    • Claim:
      10. The method of claim 9, wherein the tetrazine is selected from the following compounds: [chemical expression included]
    • Claim:
      11. The method of claim 1, wherein the inverse Diels Alder reaction between the modified oligonucleotide and the modified tetrazin uses equimolar amounts of both components if the oligonucleotide is below 100 nucleotides.
    • Claim:
      12. The method of claim 1, wherein the inverse Diels Alder reaction between the modified oligonucleotide and the modified tetrazin uses the tetrazine in 2-20-fold excess if the oligonucleotide is larger than 100 nucleotides.
    • Claim:
      13. The method of claim 11, wherein the inverse Diels Alder reaction takes place between 0 and 100° C. in aqueous media.
    • Claim:
      14. The method of claim 11, wherein modified oligonucleotides in concentrations of at least 500 μM are reacted.
    • Claim:
      15. A kit for the post-synthetic modification of nucleic acids, comprising at least one of Compounds 1, 2, 3, 8, 9, and 10, and at least one of Compounds 5, 6, and 7.
    • Current U.S. Class:
      536/253
    • Current International Class:
      07
    • Accession Number:
      edspap.20130085271