INTRA versus INTERstrand Base Stacking in DNA and RNA Oligonucleotides under the aspect of RNAs inherent potential for frameshifts

Stacking interactions in the natural nucleic acids play a much more important role than has been previously recognized. Compared to hydrogen bonding forces, stacking interactions seem to be of equal significance, not only from a structural but also from a functional point of view. This project targets oligoribonucleotide stacking, with respect to the special biological process of frameshifting. Theoretical considerations (based on the author`s experience in studies of stacking interactions in alternative oligonucleotide systems) suggest the optimization of base stacking interactions as a basic functional determinant in frameshift activity. Thereby, the differentiation between INTRA and INTERstrand stacking is the key for the understanding how this optimization takes place. In particular, the synthesis of small RNA oligonucleotides containing programmed frameshift sites, the determination of thermodynamic parameters for their duplex forrnation and the analysis of the obtained data with respect to both base pairing and anticodon-codon stacking interactions of the coded versus re-coded frameshift model sites, is a primary goal. It is supposed that at least with respect to programmed +1 frameshift sites, the thermodynamic equilibrium of anticodon-codon pairing is shifted to the rer-coded pairing complex by optimization of stacking interactions. In this context, the synthesis of nucleotides for solid support synthesis with modified bases comprising an expanded p -electron system will be an equally important goal. The modification will be designed to improve stacking without perturbing the base pair hydrogen bonding pattern. Incorporation of these modified bases into RNA and DNA oligonucleotides should enhance thermodynamic stability; they will be especially applied to the analysis of frameshift sites. Moreover, an article recently published in Science implicates frameshift mutants as a possible cause of the Alzheimer and Down syndrome. The studies presented here will contribute to a better understanding of the generally encountered biological phenomenon of frameshifting (Science 1998, 279, 242-247).

Our major contribution was the development of a novel codon-anticodon pairing model. Its characteristic feature is the cyclic covalent bridging of two short oligonucleotides - the anticodon and the codon sequence - by appropriate linker units. The resulting stabilization enables the determination of thermodynamic parameters for the formation of very short double helical RNA. With respect to codon-anticodon pairing, the influence of nearby nucleotides by base stacking is easily quantified. The chemical project addressed a field of major importance in RNA biology, namely ribosomal translation. In particular, we intended to contribute towords a better understanding of the molecular details of tRNA/mRNA decoding under the special aspect of phenomena that concern the accuracy during ribosomal translation, such as frameshifting. (To rationalize such phenomena is of general importance, as for several mammalian deseases, among them are Alzheimer and Down syndrome, so-called frameshift mutants have been detected.) We have developed model compounds for the codon-anticodon interaction. These cyclic model compounds consist of two complementary triplet sequences (codon and anticodon) together with single nucleotide overhangs at the 3`- ends. These unpaired nucleotides are structurally relevant for codon-anticodon pairing as they can interfere with the core double helix and cause a significant stabilization by base stacking. The strength of our model compounds is that the thermodynamic influences of these nucleotides on the pairing properties can be easily quantified with methods commonly used in oligonucleotide chemistry. In this sense, the novel model is a major improvement and unique of its kind. So far, the only access for such data arrived from a codon-anticodon model described by H. Grosjeans and coworkers. They took advantage of tRNA dimerization which is found only for tRNAs of complementary anticodon sequences. Of general significance is moreover the new method we developed to synthezise the cyclic model compounds. This solid-phase approach is applicable for the preparation of any cylic oligoribonucleotide from 2 to 20 nucleotides. The biological significance of such short RNA cycles are documented in the literature. A further development of our research resulted from the fact that the nucleotides corresponding to the tRNA nucleotides in position 37 (at the 3`-end of the anticodon) are in more than 70% of all known cases chemically modified nucleotides. We have focused on the subgroup of methylated nucleotides and on the general question what is their impact on RNA secondary and tertiary structure. Up to date, the commonly held picture is that nucleoside modifications modulate a preexisting fold and cause conformational changes only at the local surroundings. In a comprehensive study we found that methylated nucleosides are major determinants in RNA secondary structure formation and that single nucleobase methylations can induce significant changes in the conformations (`refolding`) of more complex RNA sequences.