Email:
ibarvik@alma.karlov.mff.cuni.cz
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 [2].
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 [8]. The completion was made on the base of ab
initio calculations [3].
(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 [7]. 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 [2] - 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.
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