A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

Liu, Hong Yan; Gao, Xiaohu

The 81 bp PCR product was put into T-A cloning pCR 2.1 vector (Invitrogen). After sequencing, positive plasmids were selected and used as the template for PCR. The resulting PCR product was separated with 2% agarose gel and recovered with QIAEX II Gel Extraction Kit (Qiagen). The purified PCR product was used as the template for in vitro transcription with MEGAscriptT7 Kit (Ambion) according to manufacturer's instruction. 29 fluoro-modified pyrimidines (TriLink, San Diego) were added to replace CTP and UTP. RNA molecules generated by the transcription reaction were annealed with the sense strand of GFP siRNA (chemically synthesized with or without 59-Cy3 or FAM by IDT). The sequence is 59-(Cy3 or FAM)-CAAGCUGACCCUGAAGUUCUU-39. For annealing, the transcripted RNA and the synthetic siRNA sense strand were mixed at molar ratio 151 in duplex buffer (IDT) and incubated at 94uC for 3 min followed by slow cooling to 25uC in 1 hour. The final chimera was store at 280uC. The constructs were cloned into PET28a (1) expression vector (Novagen). The constructs for dsRBD-His 6 and dsRBD-His 18 were obtained using full-length PKR gene (clone ID 8068981) as PCR template, and the dsRBD-His 24 and dsRBD-His 30 constructs were made by grafting additional histidines to the dsRBD-His 18 plasmid using PCR. The restriction enzyme sites for BamH1 and Xho1 were introduced in the PCR primers for cloning. dsRBD-His 6 construct was introduced with two stop codons (TAA and TGA) before the Xho1 site. For the other three constructs, the reading frames cover the His 6 sequence in the vector at the C-terminal end before the stop codon. The PCR products and PET28a (1) Single colonies were selected and grown at 37uC for 12 h in Circlegrow medium containing 30 mg/ml kanamycin. Overnight cultures were diluted at 15100 (v/v) into fresh medium and incubated at 37uC until the OD 600 values reach 0.5-1.0. Expression was induced by addition of isopropyl-b-D-thiogalactopyranoside (IPTG, 1 mM), and cell growth was continued for another 4-5 hour at 30uC. Cells were harvested by centrifugation (Beckman JA-10 rotor) at 10,000 g for 10 min and stored at 220uC.