Evaluation of new linkers and synthetic methods for internal modified oligonucleotides

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.