Conjugation of 5′‐Functionalized Oligodeoxyribonucleotides with Properly Functionalized Ligands
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- Abstract
- Table of Contents
- Materials
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- Literature Cited
Abstract
This unit reports the synthesis of oligodeoxyribonucleotides covalently linked via their 5? termini to various ligands such as intercalating agents, reactive groups, or labels. Methods for incorporation of halogenoalkyl, isothiocyanate, and 2?pyridyldisulfide linkers onto ligands, and addition of amino, carboxyl, thiophosphate, phosphate, and masked thiol groups at the 5 terminus of an oligodeoxyribonucleotide are described elsewhere in the series. This unit reports procedures for coupling the ligands and oligonucleotides, as well as details for purification and characterization of the final products.
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- Basic Protocol 1: Conjugation of 5′‐Carboxylated Oligodeoxyribonucleotides to Aminoalkylated Ligands
- Basic Protocol 2: Conjugation of 5′‐Aminoalkylated Oligodeoxyribonucleotides to Ligands Functionalized with an Isothiocyanate or N‐Hydroxysuccinimidyl Group
- Basic Protocol 3: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphorothioates to Ligands Functionalized with Halogenoalkyl, 2‐Pyridyldithio, or Iodoacetamidyl Groups
- Basic Protocol 4: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphates to Ligands Functionalized with Amino Groups
- Basic Protocol 5: Conjugation of 5′‐Mercaptoalkylated Oligodeoxyribonucleotides to Ligands Functionalized with Halogenoalkyl, Iodoacetamidyl, or 2‐Pyridyldithio Groups
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1: Conjugation of 5′‐Carboxylated Oligodeoxyribonucleotides to Aminoalkylated Ligands
Materials
Basic Protocol 2: Conjugation of 5′‐Aminoalkylated Oligodeoxyribonucleotides to Ligands Functionalized with an Isothiocyanate or N‐Hydroxysuccinimidyl Group
Materials
Basic Protocol 3: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphorothioates to Ligands Functionalized with Halogenoalkyl, 2‐Pyridyldithio, or Iodoacetamidyl Groups
Materials
Basic Protocol 4: Conjugation of Oligodeoxyribonucleotide 5′‐Phosphates to Ligands Functionalized with Amino Groups
Materials
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Figure 4.10.1 Conjugation of 5′‐carboxylated oligodeoxyribonucleotides with aminated ligands. Abbreviations: B, base (i.e., thymine, cytosine, adenine, or guanine); EDC, 1‐[3‐(dimethylamino)propyl]‐3‐ethylcarbodiimide hydrochloride; LY, Lucifer Yellow (R). View Image -
Figure 4.10.2 UV‐visible spectrum of the Lucifer Yellow–oligodeoxyribonucleotide conjugate LY‐NH‐CO(CH2 )5 ‐NH‐CO‐d[CTCTCGCACCCATCTCTC] recorded in water between 230 and 520 nm. View Image -
Figure 4.10.3 Conjugation of 5′‐aminoalkylated oligodeoxyribonucleotides with ligands functionalized with isothiocyanate or N ‐hydroxysuccinimidyl groups. Abbreviations: OP, orthophenanthroline; OR, Oregon Green; PY, 1‐pyrenepropyl. View Image -
Figure 4.10.4 UV spectrum of the orthophenanthroline‐oligodeoxyribonucleotide conjugate OP‐NH‐C(S)‐NH(CH2 )5 ‐NH‐CO‐d[CCGCTTAATACTGA] recorded in water between 230 and 350 nm. View Image -
Figure 4.10.5 UV‐visible spectrum of the Oregon Green–oligodeoxyribonucleotide conjugate OR‐NH‐C(S)‐NH(CH2 )5 ‐NH‐CO‐d[CCGCTTAATACTGA] recorded in water between 230 and 600 nm. View Image -
Figure 4.10.6 UV‐visible spectrum of the pyrene‐oligodeoxyribonucleotide conjugate PY‐C(O)‐NH‐(CH2 )6 ‐NH‐CO‐d[CCGCTTAATACTGA] recorded in water between 230 and 400 nm. View Image -
Figure 4.10.7 Conjugation of oligodeoxyribonucleotide 5′‐phosphorothioates with halogenoalkylated ligands (left), an iodoacetamidylated ligand (right), and a 2‐pyridyldithioalkylated ligand (center). For halogenoalkylated ligands, R1 = Acr‐NH‐(CH2 )6 ‐ ( UNIT , structure 1e); Pso‐(CH2 )6 ‐ ( UNIT , structure 2f), OP‐C(O)‐(CH2 )5 ‐ ( UNIT , structure 3c); and TO‐(CH2 )8 ‐ ( UNIT , structure 4e). For the iodoacetamidylated ligand, R2 = FLU. For the 2‐pyridyldithioalkylated ligand, R3 = P50 ‐(CH2 )6 ‐ ( UNIT , structure 2h). Abbreviations: Acr, acridine; FLU, fluorescein; OP, orthophenanthroline; Pso, psoralen; PY , 2‐pyridyl; TO, thiazole orange. View Image -
Figure 4.10.8 UV‐visible spectrum of the acridine‐oligodeoxyribonucleotide conjugate Acr‐NH(CH2 )6 ‐S‐p‐d[CCGCTTAATACTGA] recorded in water between 230 and 530 nm. View Image -
Figure 4.10.9 UV‐visible spectrum of the psoralen‐oligodeoxyribonucleotide conjugate Pso‐(CH2 )6 ‐S‐p‐d[CCGCTTAATACTGA] recorded in water between 220 and 400 nm. View Image -
Figure 4.10.10 UV‐visible spectrum of the thiazole orange–oligodeoxyribonucleotide conjugate TO‐(CH2 )8 ‐S‐p‐d[CCGCTTAATACTGA] recorded in water between 230 and 600 nm. View Image -
Figure 4.10.11 UV‐visible spectrum of the psoralen‐oligodeoxyribonucleotide conjugate Pso‐(CH2 )6 ‐S‐S‐p‐d[CCGCTTAATACTGA] recorded in water between 220 and 400 nm. View Image -
Figure 4.10.12 UV‐visible spectrum of the fluorescein‐oligodeoxyribonucleotide conjugate FLU‐NH‐C(O)‐CH2 ‐S‐p‐d[CCGCTTAATACTGA] recorded in 0.1 M sodium bicarbonate buffer, pH 9, between 230 and 600 nm. View Image -
Figure 4.10.13 Condensation reaction of oligodeoxyribonucleotide 5′‐phosphates with aminated compounds. R, H2 N(CH2 )5 CH2 ‐. View Image -
Figure 4.10.14 Conjugation of oligodeoxyribonucleotides bearing a 5′‐(2‐pyridyldithioalkylated) linker with a halogenoalkylated ligand (top) or a 2‐pyridyldithioalkylated ligand (bottom). R1 = TO‐(CH2 )8 ‐ ( UNIT , structure 4e); R2 = Pso‐(CH2 )6 ‐ ( UNIT , structure 2h). Abbreviations: TCEP, Tris‐(2‐carboxyethyl)phosphine hydrochloride; X, I; Py, 1‐pyridyl; L, CH2 CH2 OCH2 CH2 OCH2 CH2 . View Image -
Figure 4.10.15 UV‐visible spectrum of the psoralen‐oligodeoxyribonucleotide conjugate Pso‐(CH2 )6 ‐S‐S‐CH2 CH2 ‐(OCH2 CH2 )2 ‐p‐d[CTCTCGCACCCATCTCTC] recorded in water between 230 and 400 nm. View Image
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Literature Cited
| Literature Cited | |
| Asseline, U., Bonfils, E., Dupret, D., and Thuong, N.T. 1996. Synthesis and binding properties of oligonucleotides covalently linked to an acridine derivative: New study of the influence of the dye attachment site. Bioconjugate Chem. 7:369‐379. | |
| The authors would like to express their appreciation to past and present collaborators for their contribution to the development of varied families of oligonucleotide conjugates over the past years. This work was supported by Rhône‐Poulenc, Agence Nationale de Recherche contre le SIDA, and bioMérieux. | |
| Aubert, Y., Bourgerie, S., Meunier, L., Mayer, R., Roche, A.‐C., Monsigny, M., Thuong, N.T., and Asseline, U. 2000. Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5′‐protected thiol function and a 3′‐amino group. Nucl. Acids Res. 28:818‐825. | |
| Aubert, Y., Perrouault, L., Hélene, C., Giovannangeli, C., and Asseline, U. 2001. Synthesis and properties of triple helix‐forming oligodeoxyribonucleotides containing 7‐chloro‐7‐deaza‐2′‐deoxyguanosine. Bioorg. Med. Chem. 9:1617‐1624. | |
| Ivanovskaya, M.G., Gottihk, M.B., and Shabarova, Z.A. 1987. Modification of Oligo(poly)nucleotide Phosphomonoester groups in aqueous solutions. Nucleosides‐Nucleotides 6:913‐934. | |
| Raymond, F., Asseline, U., Roig, V., and Thuong, N.T. 1996. Synthesis and characterization of O6 modified deoxyguanosine‐containing oligodeoxyribonucleotides for triple helix formation. Tetrahedon 52:2047‐2064. | |
| Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hood, L.E. 1985. The synthesis of oligonucleotides containing an aliphatic amino group at the 5′ terminus: Synthesis of fluorescent DNA primers for use in DNA analysis. Nucl. Acids. Res. 13:2399‐2412. | |
| Thuong, N.T. and Asseline, U. 1991. Oligodeoxyribonucleotides attached to intercalators, photoreactive and cleavage agents. In Oligodeoxyribonucleotides and Analogues: A Practical Approach (F. Eckstein, ed.) pp.283‐308. IRL Press, Oxford. |
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