Intracellular Inducible Alkylation System That Exhibits Antisense Effects with Greater Potency and Selectivity than the Natural Oligonucleotide

摘要

Synthetic oligonucleotides (ODNs) that incorporate a chemically reactive appendage have been widely investigated in the endeavor to create new functions through specific reactivity toward a target sequence. A number of biotechnological approaches have been invented based on sequence-selective alkylation,[1] cross-linking,[2], [3] strand cleavage,[4]-[7] chemical ligation,[8] signal amplification,[9] and other techniques. Cross-linking oligonucleotides have been used to stabilize complexes with the cellular target mRNA by covalent-bond formation, and have been shown to be useful in enhancing antisense effects. Emerging knowledge about the fundamental role of micro-RNAs (miRNAs) in gene regulation has led to great interest in efficient antisense oligonucleotides against miRNAs as a tool for specifically knocking down particular miRNAs.[10], [11] Psoralen derivatives are activated by UV irradiation and are the only reagents applicable to in vitro and in vivo studies. They not only enhance antisense effects[12], [13] but also induce specific mutations at the sites of the reaction within the triplex DNA.[14], [15] However, until now the use of alkylating oligonucleotides in biological studies has been hampered by the lack of an efficient cross-linking agent. As alkylating or cross-linking agents tend to be unstable under physiological conditions, the use of stable precursors with inducible reactivity was expected to provide a solution to this problem.[2], [16] We recently proposed a new concept of inducible reactivity, in which a phenylsulfide derivative used as a stable precursor to a 2-amino-6-vinylpurine nucleoside analogue is automatically activated in the proximity of the target cytidine residue to form a covalent bond selectively (Scheme 1).[17]-[20] The selectivity of this inducible alkylation system for cytidine is so high that a single nucleotide difference can be discriminated in the alkylation. Herein we describe the capacity of the inducible alkylation system to exhibit antisense effects in an intracellular environment with greater potency and selectivity than the corresponding unmodified oligonucleotide.