A systematic analysis is presented of the ESR spectra from DNA oligomers of a range of sizes, including a polymer, spin-labeled with nitroxide moieties attached by different tethers. The complexity of the DNA dynamics is dealt with by the use of the general slowly relaxing local structure (SRLS) model, wherein the nitroxide moiety is reorienting in a restricted local environment, which itself is relaxing on a longer time scale. The slower motion describes the global tumbling of the DNA lattice, and the faster motion, the internal dynamics. In the present analysis, the correlation times for the axially symmetric global tumbling were those obtained from hydrodynamic theory, while the correlation times for the internal dynamics and the order parameter, which directly measures its restricted range of motion, were determined by nonlinear least-squares fits to the spectra. The principal result is the observation and characterization of two types of spectra from these labeled DNA systems. These two spectra represent components that differ from each other with respect to their local environments, one a highly restricted site yielding a large order parameter (0.61) and slower internal motions and the other a much less restricted site (with order parameter of 0.18) and faster internal motions. Whereas the one-atom tethered DUTA exhibits both sites (in roughly 9:1 ratio with the more restricted site more prevalent), the two-atom tethered DUMTA and five-atom tethered DUAT exhibit just the more restricted site, but the five-atom tethered DUAP exhibits only the less restricted site. (DUAP differs from DUAT and the others in having a less flexible tether.) It is suggested that the spin labels trapped in the highly restricted/slow motional site have a stronger interaction with the base than those in the other site. In general, the longer the tether, the faster are the correlation times for internal dynamics. For all tethers, it is found that the correlation time for internal motion perpendicular to the internal symmetry axis systematically becomes slower as the size of the oligomer increases. It is suggested that this may be a manifestation of collective modes of motion of the DNA. It is pointed out that the simpler models used in previous ESR studies are simplified cases of the more realistic SRLS model.

A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonucleotide probe, HEL878F, designed to be complementary to the nucleic acid sequence 878-896, a highly variable segment of the 16S ribosomal RNA of E. hellem spores. The specificity of this probe for its ribosomal RNA target site was confirmed using RNA degradation, ribosomal RNA target site competition, and nucleotide base mismatch control probe assays. Furthermore, the specificity of the HEL878F oligonucleotide probe for E. hellem spores was established when it was evaluated on spores from all three species of the genus Encephalitozoon that had been seeded in reagent water and environmental water concentrates. The specificity of the HEL878F oligonucleotide probe was further corroborated when tested on algae, bacteria, and protozoa commonly found in environmental water. The study demonstrates the applicability of a fluorescent in situ hybridization assay using a species-specific fluorescent-labeled oligonucleotide probe for the detection of E. hellem spores in water samples.

DNA motions consist of several components which couple, making their investigation difficult. This study describes an approach for obtaining dynamical information by EPR when spin-labeled nucleic acids are examined. The analysis is accomplished by implementing two motional models. The first model (i.e., dynamic cylinder model) views the spin-labeled helix as a diffusing cylinder containing internal dynamics which are characterized by an order parameter. The second model (i.e., base disk model) provides correlation times describing the diffusion of the spin-labeled base. In each model, the nitroxide motion consists of both global and internal contributions. Dynamic cylinder and base disk simulations of four duplexes containing nitroxides attached to thymidine by a two-atom tether (DUMTA)-(dT)7DUMTA-(dT)7.(dA)15, [(dT)7DUMTA(dT)7]2.(dA)30, [(dT)7DUMTA(dT)7]3.(dA)45, and [(dT)7DUMTA(dT)7]m.-(dA)n--demonstrate the useful application of this approach. From dynamic cylinder simulations, the order parameter for internal motions is found to be independent of the helix length (S = 0.32 +/- 0.01). Previous base disk simulations of a DNA 26mer and polymer labeled with a five-atom-tethered nitroxide seemed to indicate that tau perpendicular was only sensitive to internal dynamics. Results from base disk simulations of DUMTA-labeled DNA indicate that the perpendicular component of the base disk correlation time (tau perpendicular = 1.4-6.2 ns) is sensitive to global dynamics. Thus, tau perpendicular is a quantitative indicator of both internal and global dynamics. Comparison of the two models reveals that tau perpendicular infinity S2 tau rb, where tau rb represents the rigid-body diffusion of the DNA helix. This relationship between S and tau perpendicular provides a framework for studying conformational changes and size-dependent phenomena in spin-labeled nucleic acids. Application of the dynamic cylinder model to a B-Z transition generates distinct values of S for each of the conformations, indicating that Z-DNA is more rigid than B-DNA.

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We have used site-specifically spin-labeled polyribonucleotides to extract motional information for single-and double-stranded polyribonucleotides. The spin-label is attached to either position 4 or 5 of an uridine analogue, and in the latter position the label is linked through various tethers to the base.