Evaluation of New Linkers and Synthetic Methods for Internal Modified Oligonucleotides
| Autor: | Ronald M. Cook, Troy A. Walton, Daren J. Dick, Matthew H. Lyttle |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Pharmacology
Base Sequence Rhodamines Oligonucleotide Organic Chemistry Temperature Biomedical Engineering Pharmaceutical Science Bioengineering Nucleic Acid Denaturation Polymerase Chain Reaction Combinatorial chemistry Structure-Activity Relationship chemistry.chemical_compound Cross-Linking Reagents Förster resonance energy transfer Oligodeoxyribonucleotides chemistry Fluorescence Resonance Energy Transfer Organic chemistry Oligonucleotide Probes DNA Fluorescent Dyes Biotechnology |
| Zdroj: | Bioconjugate Chemistry. 13:1155-1158 |
| ISSN: | 1520-4812 1043-1802 |
| DOI: | 10.1021/bc0200125 |
| Popis: | Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine. The products were compared in functional assays. They performed very similarly both in a fluorescence-based melting point assay as well as in quantitative PCR. Another set of beta-actin probes were synthesized utilizing N4 [N-2-(ethylene glycol ethyl)-5-methyl]cytidine and solid-phase Tamra amidite addition at positions flanking those of the uridine. These versions gave lower T(m)s than either uridine-labeled probe and did not work as well in quantitative PCR. A control experiment using oligonucleotides with the same modified residues but without fluorophores attached revealed the same trend as the T(m) study of internal Tamra-labeled probes. Experimental details for the synthesis, purification, and testing are presented. |
| Databáze: | OpenAIRE |
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