Kundu Mrinalkanti, Nagatsugi Fumi, Majumdar Alokes, Miller Paul S, Seidman Michael M
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Nucleosides Nucleotides Nucleic Acids. 2003 Oct;22(10):1927-38. doi: 10.1081/NCN-120025240.
Reagents that recognize and bind specific genomic sequences in living mammalian cells would have great potential for genetic manipulation, including gene knockout, strain construction, and gene therapy. Triple helix forming oligonucleotides (TFOs) bind specific sequences via the major groove, but pyrimidine motif TFOs are limited by their poor activity under physiological conditions. Base and sugar analogues that overcome many of these limitations have been described. In particular, 2'-O-modifications influence sugar pucker and third strand conformation, and have been important to the development of bioactive TFOs. Here we have analyzed the impact of 2'-O-hydroxyethyl (2'-HE) substitutions, in combination with other 2' modifications. We prepared modified TFOs conjugated to psoralen and measured targeting activity in a gene knockout assay in cultured hamster cells. We find that 2'-HE residues enhance the bioactivity of TFOs containing 2'-O-methyl (2'-OMe) modifications, but reduce the bioactivity of TFOs containing, in addition, 2'-O-aminoethyl (2'-AE) residues.
能够识别并结合活的哺乳动物细胞中特定基因组序列的试剂，在基因操作方面具有巨大潜力，包括基因敲除、菌株构建和基因治疗。三链螺旋形成寡核苷酸（TFO）通过大沟结合特定序列，但嘧啶基序TFO在生理条件下活性较差，受到限制。已描述了克服这些限制的许多碱基和糖类似物。特别是，2'-O-修饰影响糖的构象和第三链的构象，对生物活性TFO的开发很重要。在这里，我们分析了2'-O-羟乙基（2'-HE）取代与其他2'修饰相结合的影响。我们制备了与补骨脂素偶联的修饰TFO，并在培养的仓鼠细胞的基因敲除试验中测量了靶向活性。我们发现，2'-HE残基增强了含有2'-O-甲基（2'-OMe）修饰的TFO的生物活性，但降低了另外含有2'-O-氨乙基（2'-AE）残基的TFO的生物活性。
