Rnase inhibitor

AuNPs (20 and 40 nm) were obtained from BBI Solutions (Cardiff, UK). All oligonucleotides (Probe-A, Probe-B, ssDNA-15, ssDNA-20, agDNA-15, agDNA-20, RNA-15, NA, NB, and MB) shown in Figure S1 were purchased from FASMAC (Kanagawa, Japan). T7 RNA polymerase was purchased from Takara Bio Inc. (Shiga, Japan). 96-7 DNA polymerase and RNase inhibitor were purchased from Nippon Gene (Tokyo, Japan). Deoxynucleotide solution mix (dNTPs) and ribonucleotide solution mix (rNTPs) were purchased from New England Biolabs Inc. (Ipswich, ME, USA). Human α-thrombin was purchased from Funakoshi (Tokyo, Japan). Transferrin, human immunoglobulin G (IgG), human serum albumin (HSA), and bovine serum albumin (BSA) were purchased from Merck (Munich, Germany). NAP-5 columns were provided with the oligonucleotides. Absorption spectra and fluorescence intensities were measured using microplate reader (TECAN, Zürich, Switzerland). All photographs were taken using a digital camera (SONY, Tokyo, Japan).

The protocells were prepared based on the inverted emulsion methods described by K. Nishimura et al.^{8 (link)}. POPC and cholesterol dissolved in chloroform (100 mg/ml) were mixed in a 9:1 weight ratio in 400 µl paraffin, and water in oil emulsion was made by vortex for 30 s with 20 µl PUREfrex 1.0 system reaction mixture including 10 ng/µl template DNA, 1 µl (40 unit) of RNase inhibitor (Nippon Gene, Toyama, Japan) and 330 mM sucrose. After layering the emulsion (400 µl) on 150 µl of outer buffer (100 mM HEPES, 280 mM potassium glutamate, 20 mM Mg(OAc)₂, NTPs (3.75 mM ATP, 2.5 mM GTP, 1.35 mM CTP, 1.35 mM UTP), amino acids (0.3 mM Tyr, 0.3 mM Cys and 0.375 mM for other 18 amino acids), 15 mM creation phosphate, 330 mM glucose, 20 µg/ml RNase; pH = 7.6) in a microtube, protocells were formed by centrifuge at 18,000 × g for 30 min at 4 °C. Outer buffer containing protocells was collected by piercing the bottom of the tube using a syringe with 18 G × 1 1/2 inch needle.