19th-banner-rev.gif

Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Influence of Na+ and Mg2+ Ions on Thermal Stability of Oligonucleotide Duplexes

Oligonucleotides and their derivates are widely used as universal molecular tools in different fields of gene diagnostics and molecular biology. In most cases the applicability of oligonuleotides depends on their hybridization ability, which is the composite function of oligonucleotide sequence, their modifications and environment. For native oligonuleotides the accurate prediction of thermal stability of various duplexes, perfect and imperfect ones, is available in the review (1). To date errorless calculations of melting temperatures of duplexes are possible for limited types of aqueous solutions containing high salt concentration at standard conditions: 1M NaCl, neutral pH (1) or various concentrations of the monovalent cation (2). Most of the models based on Poisson-Boltzmann Theory (PB) (3), Counterion Condensation Theory (CC) (4) or semiempirical relationship (2, 5) allowed the qualitative or quantitative description of the duplex stability vs ionic strength of solution containing only one type of cation. Problems of prediction of the melting temperature values appeared also when concentration of salt vary in wide range [small ligand binding (SLB) model (for example see, 6)] or upon the competitive binding of different types of positively charged ligands to an oligonucleotide duplex. PB and CC theories as distinguishing from all others can qualitatively describe the thermal stability of polymeric dsDNA in the case of the competitive binding of two types of the cations. To predict oligonucleotide duplex stability the semiempirical model was applied (5). This model considers the multivalent cation as the some additional equivalent to the total concentration of monovalent cations. So, up to now it is difficult to predict stability of oligonucleotide duplexes on the basis of non-empirical model in conditions of a composite buffer.

The goal of this work was a development of the model describing the influence of buffer composition (Na+ and Mg2+) on thermal stability of complexes formed by native and locally modified oligonucleotides. The bridged oligonucleotides containing non-nucleotide insert in sugar-phosphate backbone were used as the modified oligomers with downregulated hybridization properties (7, 8). This type of derivatives we suggested earlier as sequence specific probes. We have studied the thermal stability of DNA/DNA complexes in the wide range of cation concentration [(Na+): 0.01- 1.02 M; (Mg2+): 0.5-100 mM]. The UV melting technique was used to obtain hybridization characteristics of unmodified and bridged oligonucleotides containing the inserts on the basis of diethylene glycol (7). The database of thermodynamic parameters of duplex formation was obtained. It contains 407 experimental characteristics (including 191 ones for modified duplexes) in addition to 535 items presented in the literature. The duplex length varied from 6 to 30 base pairs.

Using this data set, we have developed the model for calculation of stability of complexes formed by native and bridged oligodeoxynucleotides with complementary strands in case of competitive binding of Na+ and Mg2+ to DNA. The basic presuppositions of the model are presented on Scheme. We proceed from the small ligand binding model (6) and took into account the possibility of non-specific condensation of cations on single stranded forms of interacting oligomers upon the increase of ionic strength. In addition to that we proposed, for the first, that the binding of Na+ and Mg2+ to any phosphate residue in DNA is independent, and for the second, that the affinity of Na+ and Mg2+ cations to ssDNA is different. The main assumption is that the higher ionic strength of solution, the lower number of additional (thermodynamically associated) counterions binds to DNA upon duplex formation.


Unlike the well-known SLB model the proposed scheme allowed us to describe the nonlinear dependence of melting temperature for all considered duplexes on logarithm of salt concentration in the presence of single type of the cation (Na+ or Mg2+). Moreover, this model properly predicts melting temperatures in the cases when Na+ and Mg2+ competitively binds to oligonucleotide species. The average error of the calculated Tm value is less than 2 °C.

Acknowledgements

This work was supported by integration grants of SB RAS (55, 73), by Program MCB of RAS, by CRDF (RUX0-008-NO-06, Y2-B-08-03) and RFBR (06-04-49263).

References and Footnotes
  1. SantaLucia, J. Jr., Hicks, D. Annu Rev Biophys Biomol Struct 33, 415-440 (2004).
  2. Owczarzy, R., You, Y., Moreira, B. G., Manthey, J. A., Huang, L., Behlke, M. A., Walder, J. A. Biochemistry 43, 3537?3554 (2004).
  3. Korolev, N., Lyubartsev, A. P., Nordenskiold, L. Biophys J 75, 3041-3056 (1998).
  4. Manning, G. S. Q. Rev Biophys 11, 179-246 (1978).
  5. von Ahsen, N., Wittwer, C. T., Schutz, E. Clinical Chemistry 47, 1956-1961 (2001).
  6. Akhrem, A. A., Lando, D. Yu. Mol Biol (Mosk) 13, 1098-1109 (1979).
  7. Pyshnyi, D. V., Lomzov, A. A., Pyshnaya, I. A., Ivanova, E. M. J Biomol Struct Dyn 23, 567-580 (2006).
  8. Lomzov, A. A., Pyshnaya, I. A., Ivanova, E. M., Pyshnyi, D. V. Dokl Biochem Biophys 409, 211-215 (2006).

Alexander A. Lomzov
Dmitrii V. Pyshnyi*

Institute of Chemical Biology and Fundamental Medicine
Siberian Branch of the Russian Academy of Sciences
Prosp. Lavrenteva, 8
630090, Novosibirsk, Russia
Novosibirsk State University
St. Pirogova, 2
630090, Novosibirsk, Russia

*Phone: +7 383 335 62 75
Fax: +7 383 333 36 77
Email: pyshnyi@niboch.nsc.ru