OLIGONUCLEOTIDE ANALOGUES: FROM SUPRAMOLECULAR PRINCIPLES TO BIOLOGICAL PROPERTIES

Transcription

1 PL1 Oligonucleotide Analogues 21 OLIGONUCLEOTIDE ANALOGUES: FROM SUPRAMOLECULAR PRINCIPLES TO BIOLOGICAL PROPERTIES Damian ITTIG, Dorte RENNEBERG, David VONLANTHEN, Samuel LUISIER and Christian J. LEUMANN* Department of Chemistry & Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland; Chemically modified oligonucleotides show great potential for applications in DNA diagnostics, as tools in molecular biology and as antisense agents and gene silencers in functional genomics and in human therapy. Among the promising analogues is tricyclo-dna, a conformationally constrained DNA analogue. We show here recent improvements in the synthesis of the tricyclo-dna building blocks for automated oligonucleotide synthesis. Furthermore we show that also tc-dna gapmers can efficiently be synthesized. We found that a tc-gap-18-mer with a window of 8 DNA-units in the center, in complex with complementary RNA, efficiently elicits RNaseH activity. In addition such gapmers are completely stable in human or murine serum over a time period of at least 24 h. INTRODUCTION Oligonucleotides have found widespread interest and applications in DNA and RNA diagnostics, as tools in molecular biology, and as antisense agents and gene silencers in functional genomics and human therapy. For many of these applications, however, unmodified oligonucleotides are of limited use due to their insufficient biostability, bioavailability and due to the fact that their affinity to complementary DNA and RNA is in many instances not sufficient to generate a biological response. Some of these drawbacks can be overcome by chemical modification of oligonucleotides 1. All of the three components of the repetitive structural unit of DNA or RNA, namely the sugar, the phosphate and the nucleobase, are equally amenable for chemical modifications to address the mentioned drawbacks. For example replacement of the phosphate unit by a thiophosphate group leads to increased biostability and has been widely investigated in the past as first generation antisense agents in functional genomics and therapy 2. Chemical modification of the 2 -OH function of ribonucleosides as an example for sugar modification has been of interest as a means to increase biostability and RNA affinity, and has lead to the second generation of antisense oligonucleotides 3. In recent years, more complex structural variations of the underlying nucleoside unit were investigated which lead to third generation antisense oligonucleotides displaying very high RNA affinity, biostability and in some cases also improved bioavailability compared to

2 22 Ittig, Renneberg, Vonlanthen, Luisier, Leumann: unmodified DNA or RNA. Selected third generation antisense oligonucleotides are displayed in Fig. 1. FIG. 1 Selected third generation antisense oligonucleotides with improved RNA binding properties and increased biostability In our own work we concentrated in the last years on the development of tricyclo (tc)-dna (Fig. 1). As LNA, tc-dna belongs to the class of conformationally constrained oligonucleotide analogues. These were specifically designed to increase complementary RNA or DNA affinity by reducing the entropy change upon duplex formation via structural preorganization of the single strand. We reported in the past on the synthesis and duplex formation properties of tricyclo-dna 4,5 as well as on its use as antisense oligonucleotide in cellular assays. Here we report now on recent advances in the synthesis of tricyclo-dna as well as on the chemical and biochemical properties of tc-dna/dna/tc-dna gapmers. RESULTS AND DISCUSSION We recently improved the original synthesis of tricyclo-dna by developing a new route to the central intermediate 1 from D-mannose in 10 steps (Fig. 2) 6. In a subsequent set of transformations the furanose ring is elaborated onto 1 yielding 2 in 46% over 5 steps. Bicyclo sugar 2 is then transformed in a further string of three steps into tricyclo sugar 3 in 43% yield overall, which serves as the central intermediate for the synthesis of the nucleosides and the corresponding phosphoramidite building blocks for oligonucleotide synthesis. Key step in this reaction sequence is the stereospecific Simmons Smith cyclopropanation of the corresponding silyl-enol ether which is obtained from the 5 -keto bicyclonucleoside. With

3 Oligonucleotide Analogues 23 this methodology we can now produce intermediate 3 in g quantities in a standard laboratory setup. FIG. 2 Improved synthetic route to tricyclo sugar 3 One of the drawbacks in 2 -deoxynucleoside synthesis in general and in tricyclo-nucleoside synthesis in particular is the lack of stereoselectivity in the nucleosidation step under Vorbrüggen conditions. In the reaction of the tricyclo sugar 3 with the N/O-protected, in situ silylated nucleobases we observe in all cases anomeric mixtures with α/β ratios of ca. 3:2, irrespective of the nature of the base. Besides the loss of material in a late step of the synthesis, there are often difficulties associated in the chromatographic separation of the anomeric mixtures posing a further technical obstacle. To circumvent these difficulties we decided to embark on a two step nucleoside synthesis starting from the enol ether 4 via NIS mediated base addition (Fig. 3). We reasoned that NIS attack from the α-face would be favored for sterical reasons. FIG. 3 Improved two-step nucleoside synthesis starting from enol ether 4 Indeed, using the persilylated pyrimidine bases thymine and N-benzoylcytosine the 2 -iodonucleosides 5 were obtained in yields of up to 81% in a remarkable stereospecificity. Only β- and no α-isomers were isolated in both cases. Subsequent removal of iodine with Bu 3 SnH went smoothely and

4 24 Ittig, Renneberg, Vonlanthen, Luisier, Leumann: resulted in the pure tricyclo-deoxynucleosides 6 again in high yield. We found that this two-step procedure leads to higher yields of 6 than the Vorbrüggen procedure in the case of the pyrimidine nucleosides. Unfortunately we were unable so far to extent this method also to the synthesis of the purine tricyclonucleosides. Further work in this area is, however, in progress. As indicated in Fig. 1, tricyclo-dna is a strong RNA binder. tc-dna/rna duplexes are thermally stabilized by 3 5 C per tc-unit relative to DNA. Thus it seemed adequate to investigate into the antisense properties of tricyclo-dna. In biological experiments we showed previously that fully modified tricyclo-dna is remarkably stable against nuclease mediated degradation with pure 3 -exonucleases and in human or murine sera. We further showed that fully modified tricyclo-dna/rna duplexes were no substrates for RNase H. Consequently no RNA degradation was observed. Given these properties it was of interest to explore the potential of tc-dna to act as a splice site modulator in cellular assays. We investigated so far splice modulation in two different models. In a first assay we showed that aberrant splicing of a mutant β-globin gene can be corrected by covering the erroneously activated 3 -cryptic splice site by an antisense oligonucleotide 7. We found that splice correction of tc-dna was fold more efficent compared to a 2 -OMe phosphorothioate of the same sequence. In another experiment we showed efficient downregulation of cyclophilin A RNA and protein by targeting an exon/intron segment of the pre-mrna with an antisense oligonucleotide, leading to multiple exon skipping. A direct comparison of tc-dna with LNA showed in this case a slightly higher efficacy of tc-dna 8. The enhanced antisense effect can not be correlated alone with target affinity as LNA binds more strongly to RNA than tc-dna. Thus, other factors as e.g. differential cellular distribution of antisense oligonucleotides as a consequence of their individual chemistries seem to play also an important part. RNase H activity leading to the degradation of the RNA is an important antisense mechanism that increases the efficacy of antisense oligonucleotides when targeted to mature mrnas. A way out of the dilemma that many chemically modified oligonucleotide analogues are unable to activate RNase H consists in the use of so called gapmers, chimaeric oligonucleotides in which both ends consist of chemically modified units while the center of the sequence contains 6 10 unmodified DNA-residues (RNase H window). We therefore decided to explore whether the gapmer concept can also be applied to tc-dna.

5 Oligonucleotide Analogues 25 For this we prepared the tc-dna gap-18-mer indicated in Fig. 4, containing a DNA window of 8 nucleotides in the center, flanked by 5 tc-dna residues each on the 5 - and the 3 -side. As a negative control we used the fully modified tc-18mer and as a positive control the pure DNA/RNA duplex. These analogues were annealed with 32 P-radiolabeled complementary RNA and the corresponding duplexes incubated with RNase H. Samples were taken in regular time intervals and the degree of RNA cleavage was followed by PAGE. As can be seen from Fig. 4, the positive DNA/RNA control shows complete cleavage of the RNA already after 2 min. On the other hand the negative control with the fully modified tc-dna/rna duplex showed no cleavage even after 16 h. Interestingly, the bands with lower mobility compared to the pure RNA control are those of the duplex which does not completely denature under the conditions of the denaturing PAGE (7 M urea). This underlines the high thermal stability of tc-dna/rna duplexes. The RNA in the tc-gapmer/rna duplex is rapidly degraded by RNase H with a comparable rate to that of the positive control. This clearly shows that tc-dna gapmers are as competent as normal DNA in eliciting RNase H activity. FIG. 4 RNase H assay of a tc-dna gap-18-mer of the sequence indicated in duplex with complementary RNA. As positive and negative controls the corresponding pure DNA/RNA and the fully modified tc-dna/rna duplexes

6 26 Ittig, Renneberg, Vonlanthen, Luisier, Leumann: In order to test whether tc-dna gapmers are also resistant towards biodegradation we incubated the tc-gap-18-mer depicted in Fig. 4 in human and fetal calf serum. After given time intervals samples were taken, ethanol precipitated and the integrity of the oligonucleotides verified by PAGE. Figure 5 shows a time course of the degradation of the tc-dna gapmer and the unmodified DNA as a control. FIG. 5 Stability of the tc-gapmer shown in Fig. 4 as a function of time in human or fetal calf serum. As comparison the corresponding unmodified oligodeoxynucleotide From Fig. 5 it becomes clear that the gapmers largely resist degradation in both human and fetal calf serum while unmodified DNA is rapidly degraded under the same conditions. Thus all the advantages of the gapmer strategy, as serum stability, RNase H activity and higher thermal stability of the corresponding duplexes with RNA do also apply to the tc-dna scaffold. Given these prerequisites it becomes now of interest to study tc-dna gapmers as antisense agents for the downregulation of mature mrnas. Work into this direction is currently underway in our laboratory. REFERENCES 1. Kurreck J.: Eur. J. Biochem. 2003, 270, Dias N., Stein C. A.: Mol. Cancer Ther. 2002, 1, Altmann K.-H., Dean N. M., Fabbro D., Freier S. M., Geiger T., Häner R., Hüsken D., Martin P., Monia B. P., Müller M., Natt F., Nicklin P., Phillips J., Pieles U., Sasmor H., Moser H. E.: Chimia 1996, 50, Steffens R., Leumann C. J.: J. Am. Chem. Soc. 1999, 121, Renneberg D., Leumann C. J.: J. Am. Chem. Soc. 2002, 124, Vonlanthen D., Leumann C. J.: Synthesis 2003, Renneberg D., Schümperli D., Leumann C. J.: Nucleic Acids Res. 2002, 30, Ittig D., Liu S., Renneberg D., Schümperli D., Leumann C. J.: Nucleic Acids Res. 2004, 32, 346.