Synthesis of Oligonucleotide Conjugates via Aqueous Diels‐Alder Cycloaddition
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- Abstract
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- Literature Cited
Abstract
The conjugation of maleimide reporter groups to 5??diene?modified oligonucleotides via aqueous Diels?Alder cycloaddition is described. Detailed basic protocols are provided for the preparation of a diene?amidite (5??diene modifier), for the attachment of a diene group to the 5??terminus of oligonucleotides, and for the conjugation of maleimide reporter groups to diene?modified oligonucleotides.
Keywords: diene?amidite; 5??diene modifier; Diels?Alder conjugation; 5??labeling of oligonucleotides
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- Basic Protocol 1: Synthesis of the Diene‐Amidite
- Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligonucleotides
- Basic Protocol 3: Labeling of A 5′‐Diene‐Modified Oligonucleotide with Tamra‐5‐Maleimide
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: Synthesis of the Diene‐Amidite
Materials
Basic Protocol 2: Synthesis of 5′‐Diene‐Modified Oligonucleotides
Materials
Basic Protocol 3: Labeling of A 5′‐Diene‐Modified Oligonucleotide with Tamra‐5‐Maleimide
Materials
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Figure 4.18.1 Preparation of diene‐amidite (S.7 ). Abbreviations: Aliquat 336, tricaprylylmethylammonium chloride; CDI, 1,1′‐carbonyldiimidazole; DIPEA, N , N ‐diisopropylethylamine; TBDMS·Cl, tert ‐butyldimethylsilyl chloride. View Image -
Figure 4.18.2 Synthesis of diene‐modified oligonucleotides. Standard synthesis cycle: deblocking (deblock solution); coupling (N‐amidite/DCI); capping (Cap A and B solutions); oxidation (oxidizer solution). N‐amidite: standard dT, dC(bz), dA(bz), and dG(ib) phosphoramidites. View Image -
Figure 4.18.3 Labeling of diene‐modified oligonucleotide with TAMRA‐5‐maleimide via aqueous Diels‐Alder cycloaddition. View Image -
Figure 4.18.4 Common oligonucleotide conjugation methods. Abbreviations: FG, functional group; MMTr, 4‐monomethoxytrityl; TFA, 2,2,2‐trifluoroacetyl. View Image
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Literature Cited
| Literature Cited | |
| Bowtell, D. and Sambrook, J. (eds.) 2002. DNA Microarrays: A Molecular Cloning Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. | |
| Goodchild, J. 1990. Conjugates of oligonucleotides and modified oligonucleotides: A review of their synthesis and properties. Bioconjugate Chem. 1: 165‐187. | |
| Hill, K.W., Taunton‐Rigby, J., Carter, J.D., Kropp, E., Vagle, K., Pieken, W., McGee, D.P.C., Husar, G., Leuck, M., Anziano, D., and Sebesta, D.P. 2001. Diels‐Alder bioconjugation of diene‐modified oligonucleotides. J. Org. Chem. 66: 5352‐5358. | |
| Letsinger, R.L., Elghanian, R., Biswanadham, G., and Mirkin, C. 2000. Use of a steroid cyclic disulfide anchor in constructing gold nanoparticle‐oligonucleotide conjugates. Bioconjugate Chem. 11: 289‐291. | |
| Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hoo, 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 sequence analysis. Nucl. Acids Res. 13: 2399‐2412. | |
| Telser, J., Cruickshank, K.A., Morrison, L.E., and Netzel, T.L. 1989. Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: Comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements. J. Am. Chem. Soc. 111: 6966‐6976. |
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