The Destabilizing Effect of Ribonuclease on the Helical Dna Structure (original) (raw)
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The sucrose gradient and native DNA S20,W, an examination of measurement problems
Biochimica et biophysica acta, 1976
Sedimentation coefficients of T7, T2H AND T4 DNA were determined with isokinetic sucrose gradients in both 0.1 M and 1 M NaCl. The s values were completely equivalent to those measured by analytical ultracentrifuge and no reduction of s20,w was observed due to the presence of sucrose (anomalous sedimentation). s20,w values are calculated on the basis of both partial specific volume (v) and apparent specific volume (0). Using the latter value s20,w molecular weight relations are derived for 0.1 M and 1 M NaCl solvents. The glucosylation of T2H and T4 DNA appears to influence s20,w in a manner disproportionate to the molecular weight added by glucose.
Pulse-polarographic analysis of nucleic acids and proteins
Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 1977
Nucleic acids and proteins were studied by means of derivative and normal pulse polarography, and d.c. and a.c. polarography in connection with the dropping mercury electrode. It was shown that natural ribonucleic acids, as transfer, ribosomal and viral RNAs yield derivative pulse-polarographic peaks; from their heights and potentials conclusions can be made about their content of ordered structure in solution, similarly as in the case of deoxyribonucleic acids studied earlier. Synthetic single-stranded polyribocytidylic acid yields a well developed peak, whereas in the double-helical complex with polyriboguanylic acid it is inactive when using either derivative pulse polarography or d.c. polarography. Well developed peaks were obtained also with albumin (a protein containing reducible --S--S--groups), while only an inflex was observed on the d.c. polarogram. Proteins were also studied in media containing cobalt (Brdi6ka's solution) or nickel and it was shown that derivative pulse polarography due to its high sensitivity and accuracy enables us to carry out the measurements even in less common media than Brdi~ka's solution. This fact could be exploited in clinical chemistry as well as in the investigation of the nature of catalytic currents of proteins. The currents of double-helical polynucleotides obtained by means of normal pulse polarography exhibit a marked dependence on the initial potential and cannot represent a reliable indicator of structural changes of biopolymers in solution. They can, however, be used in studies of the influence of the polynucleotide adsorption at different potentials on the subsequent reduction.
Isoelectric characteristics and the secondary structure of some nucleic acids
Biophysics of Structure and Mechanism, 1983
The isoelectric characteristics of some nucleic acid preparations from rat liver have been examined. 10S and 4S RNA species and SV-DNA were found to have isoelectric points of 5.2, 6.0-6.7, and 4.35 respectively. The molecular charge ratios (net negative charge/nucleotide) were calculated. Using SV-DNA as a standard, these isoelectric characteristics and charge ratios have been interpreted as indicating that the 10S and 4S RNAs have 35 and 56% of the molecules involved in secondary structure.
Zeitschrift für Naturforschung. Section C: Biosciences
The Montecarlo method is used to computer simulate a random distribution of molecular lengths generated by inducing T4 DNA fragmentation through the decay of 32P atoms introduced in the molecule. Taking into account the experimental conditions we find that the value of alpha for alkali sucrose gradients is 0.46 +/- 0.02 and does not depend on the running time. Our findings also prove that the computer simulation can be utilized to analyze sedimentation profiles of DNA molecules fragmented in vivo.
A convenient tool for studying the stability of proteins and nucleic acids
Journal of Thermal Analysis and Calorimetry, 2008
The aim of this work is to discuss the thermodynamic properties, obtained by differential scanning calorimetry (DSC), of the thermal transition of proteins and nucleic acids and to analyze these data using statistical thermodynamic relations. The denaturation of the ordered, specific structures of biological macromolecules is a cooperative process and in many cases the macromolecules undergo a two-state transition. Differential scanning calorimetry, giving direct thermodynamic information, has proved to be very useful in clarifying the energetics of macromolecule transitions and in characterizing their thermal stability. Here, various examples are discussed: i) the equilibrium thermal denaturation of ribonuclease A, a model for the use of DSC by following the temperature-unfolding of the proteins, a monomolecular transition; ii) the equilibrium thermal dissociation of a DNA double helix in two strands, an example of how DSC is used to follow a bimolecular process; iii) an example of the use of DSC for studying the melting of unimolecular and tetramolecular DNA quadruple-helices.
Thermodynamic and structural features of ultrastable DNA and RNA hairpins
Journal of Molecular Structure, 2003
Short RNA and DNA hairpins have been analysed in aqueous phase by means of UV absorption and vibrational spectroscopy in the following oligodeoxynucleotide and oligoribonucleotide sequences: 5 0 -d(GC-GAA-GC)-3 0 , 5 0 -r(CGC-GNRA-GCG)-3 0 (where N ¼ U, A, C, G and R ¼ A, G) and 5 0 -r(GCG-UGAA-CGC)-3 0 . These hairpins contain GAA triloop, GNRA and UGAA tetraloops stabilised by two or three GC base pairs in their stems. The analysis of UV absorption melting profiles allowed us to confirm the high (to very high) thermodynamic stability of these hairpins through the estimation of their melting temperature ðT m Þ: FT-IR spectra revealed the presence of N-type and/or S-type sugar puckers in the hairpins. Raman spectra at the temperatures below T m provided information on the conformations of certain nucleosides involved in the hairpins, as well as on the global conformation (A or B forms) of their stems. Raman spectra recorded as a function of temperature, are consistent with the hairpin-to-random coil conformational transitions through the breakdown of interbase H-bonds, and the loss of stacking between the bases. A discussion has been carried out on the agreement between vibrational data and those available from NMR on a few number of these hairpins. q
Journal of Physical Chemistry B, 2000
Vibrational spectra of two ribonucleosides containing pyrimidine bases, i.e., uridine and cytidine, were analyzed in the solid state at low temperature by neutron inelastic scattering (NIS) and in an aqueous solution at room temperature by Raman scattering and Fourier transform infrared absorption (FT-IR). Analysis of all these experimental spectra, measured on both native and deuterated (on labile hydrogens) species, allows a complete set of information on the vibrational modes arising from the base residues and ribose to be obtained. To interpret the vibrational spectra, a harmonic force field has been estimated for isolated ribonucleosides at the Hartree-Fock (HF) and density functional theory (DFT) levels. All these calculations have been performed by means of the Becke-Lee-Yang-Parr (B3LYP) local exchange and correlation functional (for DFT calculations) and the split valence basis sets, 631G (*) , including nonstandard polarization functions on heavy atoms (for DFT and HF calculations). The lowest energy conformers of these ribonucleosides, i.e., C3′-endo/ anti and C2′-endo/anti have been used in the vibrational mode calculations, where C3′-endo and C2′-endo refer to the N-type and S-type ribose puckering, respectively, and anti designates the orientation of base with respect to the sugar. The absence of any imaginary frequencies in the vibrational calculations confirms that the optimized geometry of the ribonucleosides well corresponds to their local mimima. The addition of the zero-point vibrational energy (ZPVE) to the electronic energy does not, however, change the energy order of the conformers. In both ribonucleosides, the C3′-endo/anti conformer has a lower energy than the C2′-endo/ anti one. It has been shown that NIS intensities calculated at the DFT/B3LYP/631G (*) level account more accurately for the characteristics of the experimental spectra. Therefore, the assignment of the observed vibrational modes has been extensively discussed in this paper on the basis of the results calculated by means of the DFT method. On the other hand, comparison between observed and calculated NIS spectra of native and deuterated species can give insight on the effect of intermolecular hydrogen bonding in the solid phase. Finally, the calculated results seem to be able to interpret the Raman marker shifts observed upon the A-Z helix transitions of poly-and oligonucleotides (C3′-endo/anti to C2′-endo/anti conformational transition of the cytosine residue), as well as those related to the UUCG tetraloop hairpin, involving C2′-endo/anti uridine and cytidine residues found in the middle positions of this highly stable tetraloop.