Ciafrè S A, Rinaldi M, Gasparini P, Seripa D, Bisceglia L, Zelante L, Farace M G, Fazio V M
Istituto Medicina Sperimentale, CNR, Rome, Italy.
Nucleic Acids Res. 1995 Oct 25;23(20):4134-42. doi: 10.1093/nar/23.20.4134.
Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules.
最近已经开发了几种使用“裸DNA”分子的基因转移技术，并且已经提出了许多利用“裸DNA”的基因治疗方案。我们通过构建硫代磷酸酯（PS-）双链DNA分子和功能性转录单元，研究了提高“裸DNA载体”稳定性的可能性，从而也提高了基因转移和表达效率。我们首先通过两条互补的35个核苷酸长的寡核苷酸退火合成了短的PS-双链DNA分子。DNA修饰酶对该分子的可及性显著降低：与正常磷酸二酯分子相比，T4连接酶和激酶活性分别降低至1/2和1/6。无论是对纯化的酶（DNase I和Bal31）还是在新鲜血清、细胞培养基或肌肉蛋白提取物中的孵育，核酸酶稳定性都有所提高。硫代磷酸酯封端的完整真核转录单元（通过用PS-引物进行Taq聚合酶扩增获得）对核酸酶攻击没有显著的保护作用。相反，通过用1、2或3个PS-dNTP替代物进行Taq扩增获得的合成转录单元“微型基因”对DNase I和Bal31核酸酶活性具有抗性。与正常磷酸二酯分子相比，由T7启动子驱动的转录效率分别为96.5%、95%和33.5%（分别有1、2或3个替代物）。
