Conformational transition within the A-form of complementary nucleic acids in solution]

Circular dichroic spectra of A-DNA in 78% ethanol and of tRNA in water and ethanol solutions have been studied at different concentrations of NaCl. An increase in the Na+ concentration from 0.5.10(-4) M to 5.10(-4) M results in a shift of the positive CD band at 264 nm of the A-DNA to a longer wavelength, 272 nm. Simultaneously, the magnitude of the 210 nm band decreases. By contrast in the case of tRNA in water solution an increase in NaCl content results in straight opposite shifts of the CD spectra. This opposite behaviour is shown to the due to a difference in ions effects in water and water-ethanol solutions, since tRNA in the ethanol solution behaves in the same way as A-DNA does in 78% ethanol. We suppose that in aqueous solution in increase in the cation concentration would stabilize the helical conformations with progressively decreasing narrow groove, i. e. more wound. At a high concentration of ethanol (60--80%) the formation of specific complex between the hydrated cations and the double-stranded regions should be taken into consideration. Thus, the hydrated cations may insert into the deep groove exerting the opposite effect of unwinding.     

A sensitive microassay of nucleic acids

A simple procedure is described for fluorometric assay of small quantities of DNA and RNA. It is highly reproducible, and sensitive to less than 10⁻⁸ g of native DNA.     

Surface enhanced IR absorption of nucleic acids from tumor cells: FTIR reflectance study

The data on surface enhanced IR absorption (SEIRA) of nucleic acids deposited on a metal substrate were obtained using FTIR in reflectance mode. A 200-400 A thick gold film on a glass plate was the metal substrate. The approximate enhancement factors of the SEIRA for different vibrations of nucleic acids in our experimental conditions were 3-5. The roughness of the Au surface was about 50 A. Application of this method to nucleic acids isolated from tumor cells revealed some possible peculiarities of their structural organization, namely, the appearance of unusual sugar and base conformations, modification of the phosphate backbone, redistribution of the H-bond net, and so forth. This method enhanced a set of the bands, which is impossible to observe in conventional IR geometry. The SEIRA spectra of the RNA from tumor cells showed more sensitivity to the grade of tumor malignancy than the spectra of the DNA. After application of the anticancer drug doxorubicin to sensitive and resistant strains, the DNA isolated from these strains had different spectral features, especially in the region of the phosphate I and II bands. As induced by anticancer drugs, the conformational changes in the DNA from resistant and sensitive cancer strains could be characterized with different levels of structure disordering.     

"External chromophores" and intensive bands in circular dichroism spectra of liquid-crystalline microphases of nucleic acids

From the analysis of CD-spectra of liquid crystalline microphases formed from DNA molecules in complexes with "external chromophores" requirements are formulated for the compounds to be used in revealing peculiarities of spatial structure of nucleic acids liquid crystalline microphases. These requirements satisfied it is possible to record the CD-spectra having intensive bands in the regions of chromophores absorption. These bands prove the helical pattern of the spatial structure of nucleic acid liquid crystalline microphase.     

IR and Raman spectroscopic studies on coulombic interaction between water-soluble porphyrins and nucleic acids

We have shown previously that the N-methylpyridyl group vibrations of water-soluble porphyrins, M(TMpy-P4), are shifted to lower frequencies (0.5-2.6 cm-1) as a result of coulombic interaction between the N(+)-CH3 group of M(TMpy-P4) and the PO2 group of a nucleic acid. We have now turned over our attention to the effect of this coulombic interaction on the PO2 group vibrations of nucleic acids. Using Fourier transform ir and Raman spectroscopy, we found that the va(PO2) at 1221 cm-1 is shifted 12 approximately 17 cm-1 to higher frequencies, whereas the vs(PO2) at 1087 cm-1 is shifted 18 approximately 26 cm-1 to lower frequencies, when DNA is mixed with M(TMpy-P4). These results indicate that the N(+)-CH3 group of M(TMpy-P4) interacts preferentially with one of the two oxygen atoms of the PO2 group of the DNA duplex.     

Comparisons between oriented film and solution tertiary structure of various nucleic acids

We have considered whether or not the tertiary structure of a biomolecule is the same in a crystal (or an oriented film) as it is in solution. A methodology has been developed for comparing polarized absorption spectra obtained from a solid-state sample with those obtained from an oriented solute to further resolve this question. An electric dichroism instrument built in our laboratory was used to measure the solution dichroism signal which, along with the ordinary solution uv absorption spectra, yields polarized absorption spectra in the directions parallel and perpendicular to the applied electric field. These were then compared to polarized absorption data from oriented films of nucleic acids to determine whether the two sets of data could be rotated into coincidence. This rotation was accomplished using a computer program based on a nonlinear programming method. Four nucleic acids were studied and the film and solution data for three of these were found to be equivalent, requiring rotation through an angle of 3°?20°, depending on film humidity, to bring them into coincidence. For the fourth sample we were unable, perhaps because of signal-to-noise ratio limitations, to find a correlation. Flow dichroism and electric dichroism data were also found to be quite similar. Thus it is clear that the induced dipole moment is along the helical axis and that the physical, hydrodynamical, and electrical axes of the nucleic acid molecules are equivalent.     

The determination of the conformational properties of nucleic acids in solution from NMR data

A program, NUCFIT, has been written for simulating the effects of conformational averaging on nuclear Overhauser enhancement (NOE) intensities for the spin systems found in nucleic acids. Arbitrary structures can be generated, and the NOE time courses can be calculated for truncated one-dimensional NOEs, two-dimensional NOE and rotating frame NOE spectroscopy (NOESY and ROESY) experiments. Both isotropic and anisotropic molecular rotation can be treated, using Woessner's formalism (J. Chem. Phys. (1962) 37, 647-654). The effects of slow conformational averaging are simulated by taking population-weighted means of the conformations present. Rapid motions are allowed for by using order parameters which can be supplied by the user, or calculated for specific motional models using the formalism of Tropp (J. Chem. Phys. (1980) 72, 6035-6043). NOE time courses have been simulated for a wide variety of conformations and used to determine the quality of structure determinations using NMR data for nucleic acids. The program also allows grid-searching with least-squares fitting of structures to experimental data, including the effects of spin-diffusion, conformational averaging and rapid internal motions. The effects of variation of intra and internucleotide conformational parameters on NOE intensities has been systematically explored. It is found that (i) the conformation of nucleotides is well determined by realistic NOE data sets, (ii) some of the helical parameters, particularly the base pair roll, are poorly determined even for extensive, noise-free data sets, (iii) conformational averaging of the sugars by pseudorotation has at most second-order influence on the determination of other parameters and (iv) averaging about the glycosidic torsion bond also has, in most cases, an insignificant effect on the determination of the conformation of nucleotides.     

Extracellular nucleic acids

Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.     

Modeling nucleic acids

Nucleic acids are an important class of biological macromolecules that carry out a variety of cellular roles. For many functions, naturally occurring DNA and RNA molecules need to fold into precise three-dimensional structures. Due to their self-assembling characteristics, nucleic acids have also been widely studied in the field of nanotechnology, and a diverse range of intricate three-dimensional nanostructures have been designed and synthesized. Different physical terms such as base-pairing and stacking interactions, tertiary contacts, electrostatic interactions and entropy all affect nucleic acid folding and structure. Here we review general computational approaches developed to model nucleic acid systems. We focus on four key areas of nucleic acid modeling: molecular representation, potential energy function, degrees of freedom and sampling algorithm. Appropriate choices in each of these key areas in nucleic acid modeling can effectively combine to aid interpretation of experimental data and facilitate prediction of nucleic acid structure.     

Incorporating residual dipolar couplings into the NMR solution structure determination of nucleic acids

NMR solution structures of nucleic acids are generally less well defined than similar-sized proteins. Most NMR structures of nucleic acids are defined only by short-range interactions, such as intrabase-pair or sequential nuclear Overhauser effects (NOEs), and J-coupling constants, and there are no long-range structural data on the tertiary structure. Residual dipolar couplings represent an extremely valuable source of distance and angle information for macromolecules but they average to zero in isotropic solutions. With the recent advent of general methods for partial alignment of macromolecules in solution, residual dipolar couplings are rapidly becoming indispensable constraints for solution NMR structural studies. These residual dipolar couplings give long-range global structural information and thus complement the strictly local structural data obtained from standard NOE and torsion angle constraints. Such global structural data are especially important in nucleic acids due to the more elongated, less-globular structure of many DNAs and RNAs. Here we review recent progress in application of residual dipolar couplings to structural studies of nucleic acids. We also present results showing how refinement procedures affect the final solution structures of nucleic acids.Copyright 2001 John Wiley & Sons, Inc.