In the search for efficient, more universal strategies in combating viral and malignant diseases, much effort has been invested in recent years in the development of novel approaches aimed at the suppression of gene expression. Antigene strategies pursue the gene targeting by triple-helix-forming oligodeoxynucleotides whereas the anti-mRNA conceptions comprise the use of artificial ribozymes and so-called antisense oligonucleotides complementary to the appropriate mRNA region.
The oligonucleotides with natural chemical composition have been, however, found as unsuitable for in vivo applications because of their insufficient resistance against nucleases. That is why numerous novel-type nucleotide analogs are designed, synthesized and tested [1-6]. The potential of so far reported pool of modifications of internucleotide linkages has been further enlarged by employing the nuclease-stable, isopolar, conformationally adjustable phosphonate bond, which, with its -O-P-C-O- ester ether moiety (in contrast to electroneutral methylphosphonate linkage with non-bridging P-CH3 group), actually does not alter the original phosphodiester so much. The isopolar phosphonate analogs of oligonucleotides can be considered as an alternative to the most exploited phosphorothioate ones. They are exceptionally stable against nucleases, hybridize with the oligomer counterparts with acceptable strength, they are not chiral, and allow for a large extent of further structural tuning to provide a rich basis to modulate the conformation of the chains. Moreover, oligonucleotides consisting of both the 3-O-P-CH2-O-5 and phosphodiester linkages are capable to elicit RNAse H activity .
This study is oriented on determination of the structural features altered as a result of variations in the modification of the oligonucleotide, and on their interpretation in terms of the impact of individual internucleotide linkage modification on the complexation properties of the oligonucleotide. The aim is to receive explanations at a molecular level of the biochemical/biological results. Nanosecond scale explicit solvent molecular dynamics simulations are used to verify deductions based on Raman spectroscopy results and to predict structural properties of those oligonucleotides, which would hardly be prepared in reality.
(i) The present work deals with phosphonate analogs of the natural phosphodiester internucleoside linkage (sometimes in conjunction with various aminoalkyl-linkers). Several double and triple helical complexes were used as model systems. The nucleic acids were surrounded by a periodic box of ~10000 TIP3P water atoms. Fully solvated trajectories were computed using the AMBER 5.0 software package. The implemented force field doesn’t contain force constants needed to describe the modified parts of the phosphonate analogs . The completion was made on the base of ab initio calculations .
(ii) It seemed advantageous to test the properties of synthetic compounds at the level of dinucleotides, which are readily obtainable with a wide variety of linkage modifications. Several model structures representing dodecamer triplex chains have been studied. Each model system consisted of two mutually antiparallel uridine dodecamers and a pseudostrand consisting of six modified A-dimers bound by Watson-Crick and Hoogsteen hydrogen bridges to the first and second rU12 strands (rU12×(ApA)6*rU12).
(iii) Hybrid (modified:unmodified) duplexes containing a central mismatch enable to test the specificity of hybridization. Influence of the oligonucleotide modification on the stability, structural features of the mismatch site, and possible creation of “fake” base-pairing was investigated utilizing model system based on 5-d(GTG ATA TGC)-3 and 5-d(GCA TAT CAC)-3 oligomers with alternated central nucleobases and modified internucleotide linkages in their vicinity.
(iv) Triplex forming oligodeoxynucleotides have attracted a great deal of attention because of their potential use in antigene therapy. In inter molecular triplexes, third strand of ODN binds to the major groove of the DNA. However, in general, the binding of a third-strand ODN to a target DNA duplex is thermodynamically weaker than duplex formation itself. Thus much effort has been made to increase the affinity of the third strand for its target. ODN analogues carrying various aminoalkyl linkers have been synthesized, some of which have been shown to increase the thermal stability of triplexes . In the present work, well known aminoalkyl linkers were tested in conjunction with various phosphonate internucleotide linkages.
(v) RNase H, an endogenous enzyme that specifically degrades target RNA in the antisense oligonucleotide/RNA hybrid duplex is an important pathway for the antisense action beside the translational arrest. RNase H hydrolyses the RNA strand in an RNA/DNA hybrid in a catalytic manner. Oligonucleotides consisting of both the 3-O-P-CH2-O-5 and phosphodiester linkages are the only compounds  - along with phosphorothioates and boranophosphates - out of hundreds of others, which are capable to elicit RNase H activity. Roots of the apparent uniqueness were interpreted by means of molecular dynamics simulations.
In acknowledgments, this work was supported by the Grant Agency of the Czech Republic (project No. 203/01/1166 and No. 202/02/D114). Results have been partially obtained using computer facilities of the MetaCentrum of the Czech Universities.
 E. Uhlmann and A. Peyman, Chem. Rev., 90 (1990) 544-584
 D. Rejman, J. Snášel, R. Liboska, Z. Točík, O. Pačes, S. Králíková, M. Rinnová, P. Kois, I. Rosenberg, Nucleosides Nucleotides & Nucleic Acids, 20 (2001) 819-823
 I. Barvík Jr., J. Štepánek, J. Bok, J. Biomol. Struct. Dyn., 19 (2002) 863-875
 I. Barvík Jr., J. Štepánek, J. Bok, Comp. Phys. Comm., 147 (2002) 158-161
 J. Hanuš, I. Barvík Jr., J. Štepánek, P.-Y. Turpin, J. Bok, I. Rosenberg, M. Petrová, Nucl. Acid Res., 29 (2001) 5182-5194
 I. Barvík Jr., J. Štepánek, J. Bok, Czech. J. Phys., 48 (1998) 409-415
 N. Atsumi, Y. Ueno, M. Kanazaki, S. Shuto and A. Matsuda, Bioorg. & Med. Chem, 10 (2002) 2933-2939
 W. D. Cornell et al., J. Am. Chem. Soc., 117 (1995) 5179-5197