Staining Properties of Selected Commercial Fluorescent Dyes Toward B- and Z-DNA.

The properties of six commonly used, commercially available, fluorescent dyes were compared in staining right-handed B-DNA and left-handed Z-DNA. All showed different degree of fluorescence turn-on in the presence of B-DNA, but very little in the presence of Z-DNA. The optimal range of dye-DNA ratios of DNA was determined. While these dyes do not provide a turn-on type probe for Z-DNA, staining between B- and Z-DNA using dyes such as SYBR Green I was shown to be useful in tracking the kinetics of conformational changes between these two forms of DNA. Finally, SYBR Green I showed unique circular dichroism patterns in 4 M NaCl that change in the presence of double stranded DNA, both in the visible and UV range.

Synchrotron radiation circular dichroism spectroscopy of oligonucleotides at millimolar concentrations.

Circular dichroism spectroscopy of nucleic acids has been traditionally performed at sample concentrations orders of magnitude lower than what occur in biological systems. While recent work from us demonstrated the flexibility of an adjustable sample cell that allowed for successful recording of CD spectra of an 18- and a 21-mer double stranded DNA sequences at around 1 mM, sample concentrations beyond 1 mM present a challenge for standard benchtop CD spectrometers. In the present work, the synchrotron radiation circular dichroism (SRCD) spectra were recorded for d(CG)9 and a mixed 18-mer double stranded DNA at 1, 5, and 10 mM in 100 mM or 4 M NaCl. SRCD of low molecular weight salmon DNA was also measured at a 10 mg/ml concentration. These results represent the first report of CD spectra of DNA samples measured at concentrations comparable to those found in the nucleus. The results suggest that dsDNA maintain very similar structures at concentrations up to tens of mg/ml, as evident by the very similar CD patterns in this concentration range. Furthermore, the SRCD allowed for the recording of CD patterns of DNA in the far UV region, which is not readily accessible by standard benchtop CD spectropolarimeters. These far UV signals appear to be quite characteristic of DNA structures and are sensitive to sample conditions.

Thermal treatment affects aptamers' structural profiles.

Using anion-exchange high performance liquid chromatography under non-denaturing conditions, the conformational flexibility of adenosine-, ampicillin-, and quinine aptamers were studied. It was found that all three aptamers showed more than one species when not subjected to thermal anneal. Addition of ligand to untreated aptamers did not significantly change the structural distribution. Upon heating followed by slow cooling, however, all three aptamers were found to exist virtually solely in one structure, presumably the partial hairpin species. It was also found that sonication of quinine aptamer, but not adenosine and ampicillin aptamer, led to its elution off HPLC as virtually a single species. These changes in conformational distribution as a result of thermal anneal or sonication were further confirmed by UV/vis and circular dichroism spectroscopy, as well as melt curves. The findings provided basis for future optimization of aptamer selection and preparation, where thermal anneal can help optimize selection efficiency and improve the consistency in the interpretation of results.

Use of anion-exchange HPLC to study DNA conformational polymorphism.

Anion-exchange chromatography carried out under non-denaturing conditions is a versatile tool to differentiate DNA conformations. In this work, the utility of this form of HPLC was demonstrated in four examples. The hairpin and duplex forms of d(CG)9 were readily resolved, which allowed for the studies of the influence of salt on the equilibrium of these two forms of secondary structures. Similarly, the minimum size of Tn in the loop region required for the sequence 5'-d(CCCAA-(T)n-TTGGG)-3' to form hairpin was established to be two nucleotides using anion-exchange HPLC and fluorescence resonance energy transfer. Furthermore, the efficiency of hybridization of partially self-complementary sequences d[(CG)6Nx] was readily monitored by non-denaturing anion-exchange HPLC. Finally, different structures adopted by quadruplex-forming sequences were resolved in the same manner.