Design of Nuclease-resistant Antisense Molecules Based on the X-ray Structures of Some Nucleases

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We have designed nuclease-resistant phosphodiester antisense molecules based on the X-ray crystalographic structures of some restriction enzymes such as Eco R1 and Bam H1. The active sites of these enzymes contain several acidic and basic amino acids residues, which activate water molecules to cleave phosphodiester bonds in DNA.Therefore, if oligonucleotides (ODNs) have certain amino function at the 4'α-position, such ODNs would not be cleaved by such enzymes due to steric and/or charge repulsion. However, to synthesize a branched-sugar nucleoside at the 4'α-position, a new method should be developed. We developed a new radical chemistry with a vinylsilyl or allylsilyl group as a temporary connecting radical acceptor tether to synthesize the required 4'α-branched thymidines. Property of phosphodiester-type ODNs containing 4'α-C-aminomethyl, -ethyl, -propyl, and-N-(2-aminoethyl) carbamoylthymidines (1, 2, 4, and 5, respectively) as a potential antisense molecule are investigated in detail. The nucleosides 1, 2, 4 and 5 were incorporated into octadecamer, [5'-d[MTMTMTMTMTMTMTMTMT]-3', where M is 5-methyl-2'-deoxycytidine], instead of T at various positions. The ODNs containing the modified nucleosides formed more stable duplexes with the complementary DNA than the corresponding unmodified ODN.These ODNs also formed stable duplexes with the complimentary RNA.The ODNs containing the modified nucleosides were significantly resistant to nucleolytic hydrolysis by both snake venom phosphodiesterase (a 3'-exonuclease) and DNase I (an endonuclease) and were also very stable in PBS containing 50% human serum. This is notable, because these consist of natural phosphodiester linkages. Furthermore, the duplexes formed by the ODNs containing the modified nucleosides and their complementary RNAs were good substrates for Escherichia coli RNase H.Thus, these ODNs were identified as a candidate for antisense molecules.