Psp73 vector

The IVT vector used for the production of Luc2 mRNA was sub-cloned from pDNAs encoding photinus pyralis luciferase (pGL4; Promega Corporation, Madison, WI, USA) and with the cDNA fragment inserted into the pSP73 vector (Promega Corporation) under the control of a T7 promoter containing 120 bps of chemically synthesized poly(d(A/T) fragments at the downstream of the cDNA region [21 (link)]. Then, the vectors were linearized with BsmBI (Figure S1A), blunted with T4 DNA polymerase, purified with gel electrophoresis, and then served as templates for IVT using the mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific, USA) to generate mRNA. The mRNAs encoding green fluorescence protein (GFP) was similarly constructed from the vectors encoding green fluorescence protein (pEGFP-1; Clontech Corporation, Mountain View, CA, USA). Prior to the experiments, all transcribed mRNAs were purified with an RNeasy Mini kit (Qiagen, Venlo, The Netherlands) and analyzed for size and purity with the Agilent RNA 6000 Nano Assay on a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) (Figure S1B).

Untagged human IL-1Ra open reading frame (ORF) sequences were purchased from Thermo Fisher Scientific (Waltham, MA, USA). To construct a DNA template for in vitro transcription, the coding region of human IL-1Ra was cloned into the pSP73 vector (Promega, Madison, WI, USA) for expression under the T7 promoter. A 120-bp poly A/T sequence was cloned into the vector downstream of the protein-coding sequence to attach a poly (A) chain to the mRNA 3′ terminal. In vitro transcription was performed on linearized pSP73-IL-1Ra-Poly(A) using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, Carlsbad, CA, USA), followed by RNA purification using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Unmodified ribonucleic acid triphosphates were used for in vitro transcription. The quantity and quality of the transcribed mRNA were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer chip-based capillary electrophoresis system (Agilent Technologies, Santa Clara, CA, USA), respectively. Finally, mRNAs encoding with luciferase 2 (Luc2) (Promega) or green fluorescent protein (ZsGreen1) (pZsGreen1-N1; Takara Bio Inc., Shiga, Japan) were prepared as described above.

The pDNA and mRNA were based on the same construction process as previously reported [6 (link),7 (link)].
pNL1.3 CMV-encoding secreted NanoLuc (secNluc pDNA) for in vitro experiments and pGL4.51 [luc2/CMV/Neo] encoding Luc2 (Luc2 pDNA) for in vivo experiments were obtained from Promega (Madison, WI, USA). Amplification of pDNA was performed in Escherichia coli DH5α strain. After isolation, the pDNA was purified using an endotoxin-free plasmid purification kit. The resulting pDNA was dissolved in Milli-Q water and stored at −20 °C until use in each experiment.
DNA templates for in vitro transcription (IVT) of mRNA encoding Gluc (Gluc mRNA) for in vitro experiments and Luc2 (Luc2 mRNA) for in vivo experiments were prepared by inserting protein expression fragments into a pSP73 vector (Promega, Madison, WI, USA) containing the T7 promoter. Prior to insertion, a 120 bp poly A/T sequence was introduced downstream of the protein-coding sequence in the pSP73 vector. This modification allowed us to produce mRNA with a 120 adenine poly(A) tail at the 3’ end by a simple IVT procedure using the pSP73-poly(A) vector. The protein expression fragment was derived from DNA encoding firefly luciferase (pGL4; Promega, Madison, WI, USA).

Enhanced GFP (EGFP) mRNA was purchased from Trilink (San Diego, CA, USA). Runt-related transcription factor 2 (Runx2) mRNA was prepared by in vitro transcription (IVT) as described previously [30 (link)]. Briefly, a flag-tagged mouse Runx2 DNA sequence, a kind gift from K. Miyazono (The University of Tokyo, Tokyo, Japan), was inserted into a pSP73 vector (Promega, Madison, WI, USA) possessing a 120 bp poly A/T sequence. IVT was performed using a mMESSAGE mMACHINE T7 ULTRA Kit (Ambion, Invitrogen, Carlsbad, CA, USA), followed by purification using an RNeasy mini kit (QIAGEN, Hilden, Germany) and spectroscopic measurement of concentration at 260 nm. Purity of mRNA was assessed spectroscopically based on ultraviolet (UV) absorption, and size of mRNA was evaluated by on-chip capillary electrophoresis using Bioanalyzer Agilent2100 (Agilent, Santa Clara, CA, USA). For pDNA construction, a sequence coding EGFP (Clontech, Palo Alto, CA, USA) and that coding a flag-tagged mouse Runx2 were inserted into pCAG-GS vectors (RIKEN, Tokyo, Japan). Notably, mRNA and pDNA were designed to possess the same protein coding sequences in both cases of EGFP (Genbank: JA532579.1) and Runx2 (RefSeq: NM_001145920.2).

mRNA was prepared through in vitro transcription (IVT) using a MEGAscript T7 Transcription Kit (Ambion, Austin, TX, USA). pDNA templates for IVT were constructed from the pAcGFP vector (Clontech, Mountain View, CA, USA) for GFP, the pORF-mEPOv18 vector (Invitrogen, Carlsbad, CA, USA) for erythropoietin, and the pCMV-XL4-Bcl2 vector (OriGene, Rockville, MD, USA) for Bcl-2. The coding region of each vector was inserted into the pSP73 vector (Promega, Madison, WI, USA) for expression under the T7 promoter. To attach a poly(-A) chain to the mRNA 3′ terminal, a 120-bp poly A/T sequence was cloned into the pSP73 vector down-stream of the protein coding sequence. For chemical modification of mRNA, 5-methyl-cytidine (5-methyl-C) and/or pseudo-uridine (ψU) (TriLink, San Diego, CA, USA) was added to the IVT reaction solution to substitute for cytidine (C) and uridine (U), respectively. mRNA prepared through IVT was purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany). pDNA for in vitro and in vivo administration was prepared by inserting the coding regions of the pAcGFP vector and the pCMV-XL4-Bcl2 vector into the pCAG-GS vector (Riken, Tokyo, Japan). Cy5 labelling of mRNA or pDNA was performed with a Label IT Nucleic Acid Labelling Kit, Cy™5 (Mirus, Madison, WI, USA). The concentration of pDNA and mRNA was determined spectroscopically at 260 nm.

Plasmid RNA temples for preparing Gaussian luciferase (gluc) and Firefly luciferase (fLuc) mRNA were prepared by inserting corresponding protein-coding sequences having 120 bp poly A/T sequence into the pSP73 vector (Promega, Madison, WI, USA). Linearized gluc and fluc plasmids were used as temples for in vitro transcription using a mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific, Waltham, MA, USA) to produce gluc and fluc mRNA. The obtained mRNA was then purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The mRNA concentration was determined by measuring the absorbance at 260 nm using a NanoDrop 3300 spectrophotometer (Thermo Fisher Scientific).

pGL4.10[luc2/SV40] was purchased from Promega (Madison, WI, USA), and pZsGreen1-N1 was purchased from Clontech (Takara Bio Inc., Shiga, Japan). mRNA was prepared by in vitro transcription (IVT) using a MEGAscript T7 Transcription Kit (Ambion, Austin, TX, USA). Unmodified ribonucleic acid triphosphates were used for the IVT. The coding region of each vector was inserted into the pSP73 vector (Promega, Madison, WI, USA) for expression under the T7 promoter. To attach a poly(-A) chain to the mRNA 3 terminal, a 120-bp poly A/T sequence was cloned into the pSP73 vector downstream of the protein-coding sequence. mRNA prepared through IVT was purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was quantified by absorbance spectrophotometry using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA quality was assessed using an Agilent 2100 Bioanalyzer chip-based capillary electrophoresis system (Agilent Technologies, Santa Clara, CA, USA).