de Almagro M Cristina, Coma Silvia, Noé Véronique, Ciudad Carlos J
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Av. Diagonal 643, E-08028 Barcelona, Spain.
J Biol Chem. 2009 Apr 24;284(17):11579-89. doi: 10.1074/jbc.M900981200. Epub 2009 Mar 3.
We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.
我们分析了多聚嘌呤发夹(PPRHs)是否具有降低基因表达的能力。这些发夹由两个反平行的嘌呤结构域组成,通过一个环相连,该环允许两个结构域之间形成Hoogsteen键,并与靶多嘧啶序列形成Watson-Crick键,从而形成三链结构。为了建立实验条件,使用人类二氢叶酸还原酶(dhfr)基因作为模型。PPRHs是针对DNA模板链设计的。用这些针对dhfr基因的模板发夹转染人类乳腺癌细胞系SKBR3,导致超过90%的细胞死亡。模板PPRHs使DHFR mRNA、蛋白质及其相应的酶活性降低。此外,还测试了针对耐甲氨蝶呤的乳腺癌细胞的DHFR PPRHs活性,并观察到高细胞死亡率。鉴于难以找到长的多嘧啶片段,我们研究了如何补偿多嘧啶靶序列中嘌呤中断的存在。测量了PPRH的稳定性,结果其半衰期出人意料地长,约为5天。最后,为了测试其应用的普遍性,使用模板PPRHs针对两个参与细胞增殖的重要基因——端粒酶和生存素,分别导致80%和95%的细胞死亡。综合我们的结果表明,反平行嘌呤发夹能够结合双链DNA的模板链并降低基因转录。因此,PPRHs可被视为一种新型的调节基因表达的分子。