Helmfors Henrik, Eriksson Jonas, Langel Ülo
Department of Neurochemistry, Stockholm University, Stockholm 10691, Sweden.
Department of Neurochemistry, Stockholm University, Stockholm 10691, Sweden.
Anal Biochem. 2015 Sep 1;484:136-42. doi: 10.1016/j.ab.2015.05.023. Epub 2015 Jun 4.
An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.
本文提出了一种改进的检测方法，用于筛选使用细胞穿透肽的短寡核苷酸的细胞内递送效率。该检测方法是对先前使用荧光素酶报告基因检测细胞穿透肽的方法的改进，因为它已从24孔板形式扩大到96孔板形式，并且不再依赖于商业采购的荧光素试剂。此外，自制的荧光素试剂可用于多种细胞系以及依赖于改变荧光素酶表达的不同检测中。为了建立新的方案，通过多个双因素实验改变三磷酸腺苷、荧光素、辅酶A和二硫苏糖醇的浓度，对荧光素试剂的组成进行了信号强度和寿命方面的优化。此外，还确定了针对短干扰RNA（siRNA）和剪接校正寡核苷酸（SCO）的细胞数量和转染时间的最佳条件。使用反向转染方法实现了siRNA和SCO的最佳转染，即在接种细胞之前，孔中已经存在寡核苷酸复合物。siRNA检测的Z'值为0.73，SCO检测的Z'值为0.71，表明这两种检测都适用于高通量筛选。
