Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization.
Sequence-specific spin-labeled oligodeoxynucleotides with conformation-sensitive electron paramagnetic resonance (EPR) signals are synthesized and examined as solution-phase nucleic acid hybridization probes. Either a proxyl or tempo ring linked to the C(5) position of deoxyuridine (dU) by a nonrigid two-atom methylamino tether is incorporated within 15-mers by phosphotriester chemistry yielding stable spin-labeled probes with distinctive EPR specific activity (AEPR) values. The AEPR is greater for a proxyl-labeled than for a tempo-labeled probe and is consistent with EPR data of enzymatically labeled 26-mers [Bobst, A. M., Pauly, G. T., Keyes, R. S., and Bobst, E. V. (1988) FEBS Lett. 228, 33-36], after normalizing for percent labeling. The spectral characteristics of the free probes and the probe/target complexes are similar to those of enzymatically spin-labeled nucleic acids containing a different nonrigid two-atom-tethered spin label [Bobst, A. M., Kao, S.-C., Toppin, R. C., Ireland, J. C., and Thomas, I. E. (1984) J. Mol. Biol. 173, 63-70]. The presence of target DNA is detected in solution by EPR spectroscopy and the assay is based on the characteristic line-shape change associated with hybridization. The EPR spectra of free and bound probe reflect little interference from changes in global dynamics of the probe, and the line-shape change upon complexation results primarily from a change in local base dynamics. The presence or absence of hybridization can be detected in a loop-gap resonator with about 1 pmol of spin-labeled 15-mer within minutes.
Strobel
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Department of Chemistry, University of Cincinnati, Ohio 45221.
Kryak
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Bobst
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Bobst
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D002850:Chromatography, Gel; D003497:Cyclic N-Oxides / Q000138:chemical synthesis* / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels / Q000138:chemical synthesis*
D003497:Cyclic N-Oxides; D007202:Indicators and Reagents; D009841:Oligonucleotides; D012997:Solvents; D013113:Spin Labels; C070253:5-(((2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)amino)methyl)-2'-deoxyuridine; C070254:5-(((2,2,6,6-tetramethyl-1-oxy-4-piperidyl)amino)methyl)-2'-deoxyuridine; D004247:DNA; D003857:Deoxyuridine