Abstract
In vivo bioavailability and delivery of nucleic acids to the site of action is a severe limitation in oligonucleotide (ON) therapeutics. Equipping the ONs with cell penetrating, homing or endosomal escape peptides can enhance specificity and/or uptake efficiencies. We describe here a general procedure for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support utilizing a novel activated alkyne containing linker which enhances the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition. Conjugation reaction is efficient in millimolar concentration and submicromolar amounts at ambient temperature. The route for POC preparation involves two subsequent conjugation steps: to solid-supported ONs containing a 5′-amino modifier (1) the triple bond donor (p-(N-propynoylamino)toluic acid (PATA), p-([2-(propynyloxy)acetamido]methyl)benzoic acid (PAMBA) or 2-(propynyloxy)acetic acid (PAA)) is first coupled and then (2) an azido-functionalized peptide is attached via a triazole linkage by copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition. The fragment-conjugated POC is released from the solid support by concentrated ammonia. The method gives high conversion of ON to the POC and only involves a single purification step after complete assembly and release from the solid support. The synthesis is flexible and designed to utilize commercially available oligonucleotide and peptide derivatives without the need for specific automated synthesizers.
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References
Akhtar S, Hughes MD, Khan A et al (2000) The delivery of antisense therapeutics. Adv Drug Deliv Rev 44:3–21
Lundin P, Johansson H, Guterstam P et al (2008) Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug Chem 19:2535–2542
Lebleu B, Moulton HM, Abes R et al (2008) Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev 60:517–529
Heitz F, Morris MC, Divita G (2009) Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br J Pharmacol 157:195–206
Borrelli A, Tornesello AL, Tornesello ML et al (2018) Cell penetrating peptides as molecular carriers for anti-cancer agents. Molecules 23:295
Jirka SMG, 'tHoen PAC, Diaz Parillas V et al (2018) Cyclic peptides to improve delivery and exon skipping of antisense oligonucleotides in a mouse model for Duchenne muscular dystrophy. Mol Ther 26:132–147
Jirka SMG, Heemskerk H, Tanganyika-de Winter CL et al (2014) Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice. Nucleic Acid Ther 24:25–36
Astakhova K, Ray R, Taskova M et al (2018) “Clicking” gene therapeutics: a successful union of chemistry and biomedicine for new solutions. Mol Pharm 15:2892–2899
El-Sagheer AH, Brown T (2012) Click chemistry - a versatile method for nucleic acid labelling, cyclisation and ligation. In: Fox KR, Brown T (eds) DNA conjugates and sensors. RSC Biomolecular Sciences
Jezowska-Herrera M, Honcharenko D, Ghidini A et al (2016) Enabling multiple conjugation to oligonucleotides using “click cycles”. Bioconjug Chem 27:2620–2628
Honcharenko M, Bestas B, Jezowska M et al (2016) Synthetic m3G-CAP attachment necessitates a minimum trinucleotide constituent to be recognised as a nuclear import signal. RSC Adv 6:51367–51373
Wenska M, Alvira M, Steunenberg P et al (2011) An activated triple bond linker enables "click" attachment of peptides to oligonucleotides on solid support. Nucleic Acids Res 39:9047–9059
Honcharenko M, Romanowska J, Alvira M et al (2012) Capping of oligonucleotides with “clickable” m3G-CAPs. RSC Adv 2:12949–12962
Jezowska M, Romanowska J, Bestas B et al (2012) Synthesis of biotin linkers with the activated triple bond donor [p-(N-propynoylamino)toluic acid] (PATA) for efficient biotinylation of peptides and oligonucleotides. Molecules 17:14174–14185
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Honcharenko, M., Honcharenko, D., Strömberg, R. (2019). Attachment of Peptides to Oligonucleotides on Solid Support Using Copper(I)-Catalyzed Huisgen 1,3-Dipolar Cycloaddition. In: Gissberg, O., Zain, R., Lundin, K. (eds) Oligonucleotide-Based Therapies. Methods in Molecular Biology, vol 2036. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9670-4_9
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DOI: https://doi.org/10.1007/978-1-4939-9670-4_9
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-4939-9670-4
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