Nitroxide-Labeled Oligonucleotides as Hybridization Probes: A Comparative Study Between Nitroxide- and Fluorescent-Labeled Probes
Using solid phase phosphoramidite DNA synthesis, oligonucleotides were spin-labeled either with a novel nitroxide-labeled phosphoramidite synthesized in our laboratory or with commercially available modified phosphoramidites containing primary amine-adopting groups. Oligonucleotides with primary amine-adopting groups were then conjugated with a proxyl nitroxide-labeled succinimidyl ester. Fluorescent-labeled oligonucleotides were synthesized similarly to the spin-labeled oligonucleotides except fluorophores were used instead of nitroxides for the labeling. Emphasis was put into developing FPLC purification protocols for the isolation of clean spin- and fluorescent-labeled oligonucleotide probes for the identification of the microsporidial parasite, Encephalitozoon hellem . Two fluorescent-based approaches, fluorescent in situ hybridization (FISH) and fluorogenic probe-based 5' nuclease PCR assays, were successfully applied for E. hellem detection. While the principles involved in detection with fluorescent-labeled oligonucleotides is known, the phenomenon of using the nitroxide radical dynamics of spin-labeled oligonucleotides for E. hellem identification was explored. The nitroxide radical dynamics is monitored by EPR and the detection relies on changes in the dynamics based on the nuclease activity of Thermus aquaticus (Taq) DNA polymerase. The Taq DNA polymerase causes the enzymatic release of nitroxide radicals from the nitroxide-labeled oligonucleotide probes under PCR conditions. This generates fast-tumbling nitroxides that are easily detected by EPR. It was found that no more than one E. hellem genome is required for its unequivocal identification by EPR with Taq DNA polymerase. In conclusion, a sensitive EPR probe-based PCR approach was developed for the identification of E. hellem spores. In principle, no more than one E. hellem spore is required for its identification, and the nitroxide-labeled oligonucleotide probes were found to be superior to their fluorescent counterparts for the following reasons: 1) only a single reporter group is required thereby simplifying the synthesis and purification of the labeled oligonucleotide; 2) no interference from bulky hydrophobic fluorescent groups during hybridization; 3) superior shelf-life of nitroxide-labeled probes as compared to the fluorescent-labeled ones; 4) in contrast to the fluorogenic probe-based 5' nuclease PCR assay, no external calibration is required with EPR-based hybridization probes due to the inherent magnetic properties of nitroxide radicals.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:dna nitroxide epr microsporidia oligonucleotide
Date of Publication:01/01/2003