Development of Oligonucleotide Based

The present thesis is based on three parts. The first and second part describe the development of two different classes of metal-ion dependent artificial ribonucleases, 2'O-methyl-ribonucleic acid based artificial ribonucleases (2'-O-MeOBANs) and peptide nucleic acid based artificial ribonucleases (PNAzymes). These studies may be regarded as a development of traditional antisense methodology. Antisense mediated inhibition of gene expression can be achieved by antisense oligonucleotide hybridization with the target mRNA causing a steric blocking or generating a substrate for the endogenous RNase H. In the case of artificial ribonucleases the catalytic transestrification unit is covalently attached to the oligonucleotide scaffold and this enables the catalytic cleavage of target mRNA without the assistance of cellular enzymes and allows for a larger variety of modifications to the oligonucleotide. The developed 2'-OMeOBANs and PNAzymes are individually tailored artificial enzymes capable of sequence selective cleavage of target RNA. The general basis for the systems is that these artificial enzymes selectively hybridize with the target RNA, which brings the catalytic group to the vicinity of the scissile phosphodiester linkage thus inducing cleavage of the RNA. Several 2'-O-MeOBANs and PNAzymes have been developed and evaluated with respect to cleavage of a model of the leukemia related M-BCR/ABL mRNA. What is special with these artificial ribonucleases is that they are designed to induce formation of a non-base paired region (bulge) in the target RNA upon binding which is advantageous since RNA bulges are more predisposed to cleavage than fully duplexed RNA. In addition, when the catalytic unit is incorporated inside the duplex region the cleavage of the target RNA creates shorter complementary fragments and catalytic turnover is provided. Particularly interesting is a new class of Cu(II) dependent PNAzymes. These are highly site selective RNA cleavers displaying multiple turnover of substrate (PNAzyme:RNA, 1:100). The fastest PNAzymes displayed half-lives down to 1⁄2 h for target cleavage, which makes these the fastest and most selective artificial ribonucleases reported so far and these can be regarded to be RNA cleaving restriction enzymes. The third part of this thesis concerns peptides that can affect hybridization of doublestranded oligonucleotides. Several different cationic peptides have been synthesized and investigated for their ability to influence the thermal melting of 2'-O-MeRNA/RNA and DNA/DNA duplexes. These cationic peptides were shown to selectively increase the thermal stability of 2'-O-MeRNA/RNA duplexes, while leaving the DNA/DNA hybrids unaffected. The dramatic difference suggest that, although electrostatic interaction plays a role, there is another major and structurally related component influencing the properties of these oligonucleotide duplexes. LIST OF PUBLICATIONS This thesis is based on the following articles and manuscripts, which are referred to by their Roman numerals: I Murtola M, Strömberg, R. 2'-O-methyloligoribonucleotide based artificial nucleases (2'-O-MeOBANs) cleaving a model of the leukemia related MBCR/ABL mRNA. ARKIVOC, 2009, (3), 84-94. II Sandbrink J, Murtola M, Strömberg R. Solid support post-conjugation of amino acids and a phenanthroline derivative to a central position in peptide nucleic acids. Nucleosides Nucleotides Nucleic Acids. 2007, 26, (10-12), 1485-9. III Murtola M, Ossipov D, Sandbrink J, Strömberg R. RNA cleavage by 2,9diamino-1,10-phenanthroline PNA conjugates. Nucleosides Nucleotides Nucleic Acids. 2007, 26, (10-12), 1479-83. IV Murtola M, Strömberg R. PNA based artificial nucleases displaying catalysis with turnover in the cleavage of a leukemia related RNA model. Org Biomol Chem. 2008, 6, (20), 3837-42. V Murtola M, Strömberg R. PNAzymes that are artificial RNA restriction enzymes. Manuscript. VI Murtola M, Zaramella S, Yeheskiely E, Strömberg R. Cationic Peptides that Increase the Thermal Stability of 2'-OMeRNA/RNA Duplexes, but that do not affect DNA/DNA hybridization. Manuscript. Additional publications Ora M, Murtola M, Aho S, Oivanen M. Hydrolytic reactions of 3'-N-phosphoramidate and 3'-N-thiophosphoramidate analogs of thymidylyl-3',5'-thymidine. Org Biomol Chem. 2004, 2, (4), 593-600. Yousefi-Salakdeh E, Murtola M, Zetterberg A, Yeheskiely E, Strömberg R. Synthesis of 8-aminoadenosine 5'-(aminoalkyl phosphates), analogues of aminoacyl adenylates. Bioorg Med Chem. 2006, 14, (8), 2653-9. Murtola M, Strömberg R. Development of 2'-O-methyloligoribonucleotide and peptide nucleic acid based artificial ribonucleases. Nucleic Acids Symp Ser. 2007, (51), 201-202. Milton S, Murtola M, Sandbrink J, Yeheskiely E, Strömberg R. Making oligonucleotide conjugates and breaking oligonucleotides. Nucleic Acids Symp Ser. 2007, (51), 61.