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

我们根据一些限制性内切酶的X-射线晶体结构设计了抗核酸酶的磷酸二酯反义分子，如Eco R1和Bam H1。这些酶的活性部位含有几个酸性和碱性氨基酸残基，可以激活水分子来裂解DNA中的磷酸二酯键。因此，如果寡核苷酸(ODN)在4‘α-位具有一定的氨基功能，那么这种ODN就不会被这种酶切割，因为空间和/或电荷排斥。然而，为了合成4‘α位置上的支链糖核苷，需要开发一种新的方法。我们开发了一种新的自由基化学，以乙烯基硅基或烯丙基硅基作为临时连接自由基受体的链条来合成所需的4‘α分支胸苷。研究了4‘-α-C-氨基甲基、-乙基、-丙基和-N-(2-氨基乙基)氨基甲酰胸腺嘧啶(分别为1、2、4和5)作为潜在反义分子的磷酸二酯型寡核苷酸的性质。核苷1，2，4和5被引入到十八聚体[5‘-d[MTMTMTMTMTMTMT]-3’，其中M是5-甲基-2‘-脱氧胞苷]，而不是T在不同的位置。与未修饰的DNA相比，含有修饰核苷的ODN与互补DNA形成了更稳定的双链，并且与互补RNA形成了稳定的双链。含有修饰核苷的ODN对蛇毒磷酸二酯酶(3‘-外切酶)和DNase I(内切酶)都有明显的抵抗作用，在含有50%人血清的PBS中也非常稳定。这一点值得注意，因为这些都是由天然的磷酸二酯键组成的。此外，含有修饰核苷的ODN与它们的互补RNA形成的双链是大肠杆菌RNase H的良好底物，因此这些ODN被鉴定为反义分子的候选者。

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.