Zycy10p3s2t

Parental minicircle (MC) vectors were purchased from System Biosciences (Cat. MN511A‐1, Palo Alto, CA). We inserted miR‐378 precursor, Lxrα (liver X receptor alpha) short hairpin RNA (shRNA), Lxrα open reading frame (ORF), or Ppargc1β ORF into the minicircle parental plasmid. A transthyretin gene (TTR) promoter was used to ensure their liver‐specific expression.16 This construct was referred to as MC‐TTR‐miR‐378, MC‐TTR‐Lxrα‐shRNA, MC‐TTR‐Lxrα, or MC‐TTR‐Ppargc1β. To prepare the minicircle, a parental minicircle vector was transformed into a special host E. coli bacterial strain ZYCY10P3S2T (Cat. MN900A‐1; System Biosciences). Minicircles were prepared based on the manufacturer’s instruction.

Generation of cccDNA-like molecules was performed as previously described (15 (link)). ZYCY10P3S2T (System Bioscience) were transformed with the parental plasmids by a heat shock of 30 s at 42°C, plated on LB agar plates containing 50 μg/ml Kanamycin (Euromedex) and incubated overnight at 37°C. A colony was picked up and amplified as starter culture in terrific broth (TB) medium containing 50 μg/ml Kanamycin during 8 h at 42°C. Bacteria were further amplified in a larger volume of TB medium without Kanamycin overnight at 42°C. Two volumes of LB medium containing 40 mM NaOH and 0.1% L-Arabinose (Merck) were then added and the culture was incubated 4 h at 32°C. cccDNA-like molecules were then extracted with the Nucleobond Xtra Midi kit (Macherey-Nagel) according to the manufacturer's instructions.

rcccDNA was produced using minicircle technology as described previously.⁴⁷ (link) In brief, HBV genome of genotype D sequence was cloned into minicircle producing plasmid containing attB and attP recombination sites (mini-HBV). Chemically competent E. coli strain ZYCY10P3S2T (System Biosciences) was transformed with the mini-HBV. Clones of successfully transformed cells were selected, and three colonies of E. coli were incubated in lysogeny broth (LB) with kanamycin at 37°C for 4 h. Next, 1 mL of the resulting cell suspension was inoculated to 200 mL of Terrific broth medium and incubated at 37°C overnight to an OD₆₀₀ of 6–8 and pH of 7.0. Thereafter, it was mixed with 200 mL of induction medium (1 N NaOH and 0.2% L-arabinose in LB) and incubated first for 3 h at 30°C, then for 1 h at 37°C. rcccDNA was isolated from the resulting bacterial pellet using a Plasmid Maxi Kit (Qiagen, Germany).

We generated an in vivo expression vector of miR-206 by cloning human miR-206 precursor into mini-circle vectors purchased from System Biosciences (Cat. MN511A-1). A transthyretin gene (TTR) promoter was inserted into the upstream of miR-206 precursor to ensure liver-specific expression of miR-206 [12 (link)]. To rule out non-specific effects of the plasmid, we generated a miR-206 mismatched-expression vector by mutating the seed region of miR-206, termed MC-TTR-miR-206-MM. We inserted the coding region of mouse Ptpn1 into a mini-circle vector and the TTR promoter was used to ensure hepatic expression of Ptpn1. This vector was referred as to MC-TTR-Ptpn1. Parental MC-TTR-miR-206 or MC-TTR-Ptpn1 vector was transformed into a special host E. coli bacterial strain ZYCY10P3S2T (System Biosciences, Cat: MN900A-1). Mini-circles were generated based on the manufacturer’s instructions.

All DNA and RNA oligos were ordered from IDT. For annealing, DNA oligos were diluted with annealing buffer [30 mM HEPES (pH 7.5); 100 mM KOAc] and annealed in a PCR machine using a program that heated the reactions to 95 °C and then decreased the temperature at a rate of 1 °C per minute to 25 °C. Oligo sequences used in this work are listed in SI Appendix, Table S1. The circular 80-nt ssDNA was generated by ligating the ends of a linear 80-nt ssDNA in the presence of a short 12-nt complementary oligo that spanned the 3′/5′ junction using an approach adapted from ref. 31 (link). The ligation product was digested by ExoI (NEB) to remove linear by-products and the splint oligo and then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) to remove the enzymes. The circular gapped dsDNA was generated by annealing the circular 80-nt ssDNA with a complementary 40-nt ssDNA that spanned 3′/5′ junction. The gapped plasmid was generated by digesting a plasmid containing three sequential BbvCI sites with Nt.BbvCI (NEB). The minicircle was purified from E. coli ZYCY10P3S2T (System Biosciences) transformed with a parental plasmid containing the minicircle sequence, 32× I-SecI sites and PhiC321 attB-attP sites. The tRNA corresponded to a mixture of yeast tRNA (Invitrogen).