Abstract
Displaying ligands in a succinct and predictable manner is essential for elucidating multivalent molecular-level binding events. Organizing ligands with high precision and accuracy provides a distinct advantage over other ligand-display systems, such as polymers, because the number and position of the ligand(s) can be accurately and fully characterized. Here we describe the synthesis of peptide nucleic acids (PNAs), which are oligonucleotide mimics with a pseudopeptide backbone that can hybridize to oligonucleotides through Watson-Crick base pair to form highly predictable and organized scaffold for organizing a ligand. The ligand(s) are covalently attached to the PNA through a squarate coupling reaction that occurs between a free amine on the ligand and a free amine appended to the pseudopeptide backbone of the PNA. In this chapter we describe the synthesis of a LKγT monomer, which ultimately yields the free amine off the backbone of the PNA, incorporation of the monomer in a PNA oligomer, and the sequential squarate coupling to conjugate the ligand.
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References
Fasting C, Schalley CA, Weber M, Seitz O, Hecht S, Koksch B, Dernedde J, Graf C, Knapp EW, Haag R (2012) Multivalency as a chemical organization and action principle. Angew Chem Int Ed Engl 51:10472–10498
Mammen M, Choi S-K, Whitesides GM (1998) Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors. Angew Chem Int Ed 37:2754–2794
Dix AV, Moss SM, Phan K, Hoppe T, Paoletta S, Kozma E, Gao ZG, Durell SR, Jacobson KA, Appella DH (2014) Programmable nanoscaffolds that control ligand display to a G-protein-coupled receptor in membranes to allow dissection of multivalent effects. J Am Chem Soc 136:12296–12303
Dix AV, Conroy JL, George Rosenker KM, Sibley DR, Appella DH (2015) PNA-based multivalent scaffolds activate the dopamine D2 receptor. ACS Med Chem Lett 6:425–429
Englund EA, Wang D, Fujigaki H, Sakai H, Micklitsch CM, Ghirlando R, Martin-Manso G, Pendrak ML, Roberts DD, Durell SR, Appella DH (2012) Programmable multivalent display of receptor ligands using peptide nucleic acid nanoscaffolds. Nat Commun 3:614
Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500
Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules. Nature 365:566–568
Datta B, Schmitt C, Armitage BA (2003) Formation of a PNA2−DNA2 Hybrid Quadruplex. J Am Chem Soc 125:4111–4118
Englund EA, Appella DH (2005) Synthesis of γ-substituted peptide nucleic acids: a new place to attach fluorophores without affecting DNA binding. Org Lett 7:3465–3467
Kohler O, Jarikote DV, Seitz O (2005) Forced intercalation probes (FIT Probes): thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection. Chembiochem 6:69–77
Kuhn H, Demidov VV, Coull JM, Fiandaca MJ, Gildea BD, Frank-Kamenetskii MD (2002) Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. J Am Chem Soc 124:1097–1103
Kohhler O, Jarikote DV, Singh I, Parmar VS, Weinhold E, Seitz O (2005) Forced intercalation as a tool in gene diagnostics and in studying DNA–protein interactions. Pure Appl Chem 77:327–339
Moustafa ME, Hudson RH (2011) An azo-based PNA monomer: synthesis and spectroscopic study. Nucleosides Nucleotides Nucleic Acids 30:740–751
Ortiz E, Estrada G, Lizardi PM (1998) PNA molecular beacons for rapid detection of PCR amplicons. Mol Cell Probes 12:219–226
Robertson KL, Yu L, Armitage BA, Lopez AJ, Peteanu LA (2006) Fluorescent PNA probes as hybridization labels for biological RNA. Biochemistry 45:6066–6074
Roy S, Tanious FA, Wilson WD, Ly DH, Armitage BA (2007) High-affinity homologous peptide nucleic acid probes for targeting a Quadruplex-forming sequence from a MYC promoter element. Biochemistry 46:10433–10443
Xi C, Balberg M, Boppart SA, Raskin L (2003) Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells. Appl Environ Microbiol 69:5673–5678
Nielsen PE, Appella DH (2014) Peptide nucleic acids: methods and protocols. Humana Press, New York
Ray A, Nordén B (2000) Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future. FASEB J 14:1041–1060
Corradini R, Sforza S, Tedeschi T, Totsingan F, Manicardi A, Marchelli R (2011) Peptide nucleic acids with a structurally biased backbone. Updated review and emerging challenges. Curr Top Med Chem 11:1535–1554
Englund EA, Appella DH (2007) Gamma-substituted peptide nucleic acids constructed from L-lysine are a versatile scaffold for multifunctional display. Angew Chem Int Ed Engl 46:1414–1418
Manicardi A, Guidi L, Ghidini A, Corradini R (2014) Pyrene-modified PNAs: Stacking interactions and selective excimer emission in PNA2DNA triplexes. Beilstein J Org Chem 10:1495–1503
Scheibe C, Wedepohl S, Riese SB, Dernedde J, Seitz O (2013) Carbohydrate-PNA and aptamer-PNA conjugates for the spatial screening of lectins and lectin assemblies. Chembiochem 14:236–250
Winssinger N (2012) DNA display of PNA-tagged ligands: a versatile strategy to screen libraries and control geometry of multidentate ligands. Artif DNA PNA XNA 3:105–108
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Shank, N., George Rosenker, K.M., Englund, E.A., Dix, A.V., Rastede, E.E., Appella, D.H. (2019). Synthesis and Application of LKγT Peptide Nucleic Acids. In: Shank, N. (eds) Non-Natural Nucleic Acids. Methods in Molecular Biology, vol 1973. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9216-4_8
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DOI: https://doi.org/10.1007/978-1-4939-9216-4_8
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