Backbone modifications for antisense oligonucleotides
Eighteen different types of backbone replacements have been investigated. The thermodynamic stability of the duplexes formed between these modified oligonucleotides and their RNA complements are summarized. Among these backbone replacements, we identified two amide derivatives which display improved properties fort their application to the antisense strategy. Some important structural and conformational elements which contribute to the thermodynamic stability of the duplexes were deduced from the melting temperatures (Tm's) data and from molecular modeling and 1H-NMR studies. Antisense oligodeoxyribonucleotides constitute a promising class of potential new therapeutical agents that would interact selectively and in a predictable manner with a target RNA (ref. 1). This interaction would occur by specific Watson-Crick base pairing between the antisense and the complementary RNA strand. The formation of the duplex would prevent the expression of a defined disease-related protein. However, the naturally occurring oligonucleotides (DNA and RNA) do not meet the criteria for potential drug candidates. Chemical modifications of the 2'-oligodeoxyribonucleotides are required to improve several of their properties. Among them, the resistance of antisense oligonucleotides towards cellular nucleases has to be substantially increased, keeping (or ideally improving) the affinity and the specificity for the complementary RNA target. The poor penetration of the 2'-oligodeoxynucleotides into cells has also to be improved. Therefore, various replacements of the naturally occurring phosphodiester backbone, which is responsible for the instability of the oligodeoxynucleotides in vivo and, at least partially, for their low ability to cross cell membranes, were recently investigated (ref. 1). Unfortunately, most of the replacements of the phosphodiester linkage leading to an increased resistance towards nucleases are also connected to a decrease in affinity for the RNA complement. So far, only very few examples of backbone replacements which are not disfavoring the formation of a duplex with RNA were reported (ref. 2, 5, 6). We summarize here our results on the backbone modifications that we incorporated into 2'- oligodeoxynucleotides (Figure 1). Initially, we proposed to replace the natural phosphodiester group by an amide as in 6 - 10, keeping four atoms between two sugar moieties (ref. 3-7). The main reasons for this choice were the novelty of the structures, the compatibility of the amide bond with the conditions for the solid phase synthesis of oligonucleotides. An amide bond should be more stable under physiological conditions than a phosphodiester bond. The overall charge reduction of the oligonucleotides containing neutral amide bonds should favor their penetration through cell membranes. The amide moiety is readily accessible by simple synthetic methods and is achiral, thereby avoiding mixtures of diastereomers in oligodeoxyribonucleotides. Moreover, our molecular modeling studies suggested that these amide modified backbones would be compatible with a duplex structure with an RNA strand. These amide modifications 6 - 10 were compared to the more rigid or more flexible backbone replacements (Figure 1) in terms of thermodynamic stability of the duplexes formed with an RNA complement. In most cases, the melting temperature (Tm) of 3 duplexes (Sequences A - E, Table 1) were measured. In Table 2 the average values for the variation of the melting temperature of the duplexes (A T,/modification) are summarized.