Abes Saïd, Turner John J, Ivanova Gabriela D, Owen David, Williams Donna, Arzumanov Andrey, Clair Philippe, Gait Michael J, Lebleu Bernard
UMR 5235 CNRS, Université Montpellier 2, Place Eugene Bataillon, 34095 Montpellier cedex 5, France.
Nucleic Acids Res. 2007;35(13):4495-502. doi: 10.1093/nar/gkm418. Epub 2007 Jun 21.
Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5-10 microM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 muM concentration of the R6Pen-PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen-PNA705 structure-function relationship have also been evaluated.
作为一种分析工具和治疗方法的开发手段，对核前体信使核糖核酸（pre-mRNA）剪接机制的序列特异性干扰受到了越来越多的关注。这需要核糖核酸酶H无活性的DNA模拟物与pre-mRNA进行序列特异性且高亲和力的结合。在这方面，肽核酸（PNA）或磷酰胺吗啉代寡核苷酸（PMO）特别适合作为空间位阻寡核苷酸。然而，与细胞穿透肽（CPP）如Tat或穿膜肽缀合的PNA或PMO进行剪接校正，需要高浓度（5 - 10 microM）的此类缀合物，除非添加溶酶体溶解剂以增加从内吞小泡的逃逸。我们专注于对现有CPP进行修饰，以寻找能够在不添加溶酶体溶解剂的情况下更有效地将剪接校正PNA或PMO递送至细胞核的肽。我们在此描述R6-穿膜肽（其中精氨酸残基被添加到穿膜肽的N端）是迄今为止在剪接校正试验中测试的所有CPP中活性最高的，在该试验中，隐蔽剪接位点的掩盖允许荧光素酶报告基因的表达。通过荧光素酶发光和逆转录-聚合酶链反应（RT-PCR）监测，在1 microM浓度的R6Pen-PNA705缀合物时发生高效且序列特异性的校正。还评估了R6Pen-PNA705结构-功能关系的一些方面。
