esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.
Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.
Xuan
Baoqin
B
Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China.
Qian
Zhikang
Z
Tan
Chang
C
Min
Taishan
T
Shen
Shuiyuan
S
Huang
Weida
W
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli; D015870:Gene Expression / Q000187:drug effects*; D015684:Gene Products, pol / Q000037:antagonists & inhibitors / Q000235:genetics; D016679:Genome, Viral; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D043244:Ribonuclease III; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014162:Transfection
D015684:Gene Products, pol; D006514:Hepatitis B Surface Antigens; C066027:P protein, Hepatitis B virus; D034741:RNA, Small Interfering; D004722:Endoribonucleases; D043244:Ribonuclease III