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Direct Transfection of Fatty Acid Conjugated siRNAs and Knockdown of the Glucose-Regulated Chaperones in Prostate Cancer Cells
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-09-21 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00580 Sunil S Shah 1 , Christopher N Cultrara 1 , Stephen D Kozuch 1 , Mayurbhai R Patel 2 , Jorge A Ramos 3, 4 , Uri Samuni 3, 4 , Jenny Zilberberg 5 , David Sabatino 1
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-09-21 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00580 Sunil S Shah 1 , Christopher N Cultrara 1 , Stephen D Kozuch 1 , Mayurbhai R Patel 2 , Jorge A Ramos 3, 4 , Uri Samuni 3, 4 , Jenny Zilberberg 5 , David Sabatino 1
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The emerging field of RNAi nanotechnology has led to rapid advances in the applications of siRNAs in chemical biology, medicinal chemistry, and biotechnology. In our RNAi approach, bioconjugation of linear, V-, and Y-shaped RNA templates were designed using a series of saturated and unsaturated fatty acids to improve cell uptake and knockdown efficacy of the oncogenic glucose regulated proteins (GRPs) in prostate (PC-3) cancer cells. An optimized HCTU-coupling procedure was developed for tagging variable saturated and unsaturated fatty acids onto the 5′-ends of linear and V-shaped RNA templates that were constructed by semiautomated solid phase RNA synthesis. Hybridization and self-assembly of complementary strands yielded linear, V-, and Y-shaped fatty acid-conjugated siRNAs which were characterized by native PAGE. CD spectroscopy confirmed their A-type helix conformations. RP IP HPLC provided trends in amphiphilic properties, whereas DLS and TEM confirmed multicomponent self-assembled structures that were prone to aggregation. Subsequently, the fatty acid conjugated siRNA bioconjugates were tested for their RNAi activity by direct transfection within PC-3 cells known to overexpress oncogenic GRP activity. The siRNA bioconjugates with sense strand modifiers provided more potent GRP knockdown relative to the antisense modified siRNAs, but to a lesser extent when compared to the unconjugated siRNA controls that were transfected with the commercial Trans-IT X2 dynamic delivery system. Flow cytometry revealed that the latter may be at least in part attributed to limited cell uptake of the fatty acid conjugated siRNAs. Nonetheless, these new constructs represent an entry point in modifying higher-order siRNA constructs that may lead to the generation of more efficient siRNA bioconjugates for screening important oncogene targets and for cancer gene therapy applications.
中文翻译：
脂肪酸共轭siRNA的直接转染和前列腺癌细胞中葡萄糖调节的伴侣的敲除。
RNAi纳米技术的新兴领域已导致siRNA在化学生物学，药物化学和生物技术中的应用迅速发展。在我们的RNAi方法中，使用一系列饱和和不饱和脂肪酸设计线性，V型和Y型RNA模板的生物缀合，以改善前列腺癌（PC- 3）癌细胞。开发了一种优化的HCTU偶联程序，用于将可变的饱和和不饱和脂肪酸标记到通过半自动化固相RNA合成构建的线性和V形RNA模板的5'端。互补链的杂交和自组装产生线性，V型和Y型脂肪酸共轭的siRNA，其特征在于天然PAGE。CD光谱法证实了它们的A型螺旋构象。RP IP HPLC提供了两亲特性的趋势，而DLS和TEM证实了易于聚集的多组分自组装结构。随后，通过直接转染已知过表达致癌GRP活性的PC-3细胞内，测试了脂肪酸缀合的siRNA生物缀合物的RNAi活性。相对于反义修饰的siRNA，具有正义链修饰剂的siRNA生物共轭物提供了更强的GRP敲低能力，但与用商业Trans-IT X2动态递送系统转染的未缀合的siRNA对照相比，其作用程度较小。流式细胞仪显示后者可能至少部分归因于脂肪酸缀合的siRNA的有限细胞摄取。尽管如此，
更新日期：2018-09-21
中文翻译：
脂肪酸共轭siRNA的直接转染和前列腺癌细胞中葡萄糖调节的伴侣的敲除。
RNAi纳米技术的新兴领域已导致siRNA在化学生物学，药物化学和生物技术中的应用迅速发展。在我们的RNAi方法中，使用一系列饱和和不饱和脂肪酸设计线性，V型和Y型RNA模板的生物缀合，以改善前列腺癌（PC- 3）癌细胞。开发了一种优化的HCTU偶联程序，用于将可变的饱和和不饱和脂肪酸标记到通过半自动化固相RNA合成构建的线性和V形RNA模板的5'端。互补链的杂交和自组装产生线性，V型和Y型脂肪酸共轭的siRNA，其特征在于天然PAGE。CD光谱法证实了它们的A型螺旋构象。RP IP HPLC提供了两亲特性的趋势，而DLS和TEM证实了易于聚集的多组分自组装结构。随后，通过直接转染已知过表达致癌GRP活性的PC-3细胞内，测试了脂肪酸缀合的siRNA生物缀合物的RNAi活性。相对于反义修饰的siRNA，具有正义链修饰剂的siRNA生物共轭物提供了更强的GRP敲低能力，但与用商业Trans-IT X2动态递送系统转染的未缀合的siRNA对照相比，其作用程度较小。流式细胞仪显示后者可能至少部分归因于脂肪酸缀合的siRNA的有限细胞摄取。尽管如此，
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