All DNA sequences used in this study were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). All the oligonucleotides are given in Table S1 (see Supplementary information Table S1 ). The HiScribe T7 High Yield RNA Synthesis Kit and Monarch RNA Purification Columns were obtained from New England BioLabs (Ipswich, MA, USA). Trizol Reagent and Dynabeads M-270 Streptavidin (magnetic beads [MBs], 2.8 µm in diameter, 10 mg/mL) were provided by Thermo Fisher Scientific (Waltham, MA, USA). Purified Leptotrichia wade (Lwa)Cas13a protein and LwaCas13a buffer were obtained from Tolo Biotech (Shanghai, China). RNase inhibitor was obtained from Promega Corporation (Madison, WI, USA). Hemin and AzBTS-(NH4)2 were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO, USA). RNase-free water and all chemicals were bought from Sangon Biotechnology (Shanghai, China) and used without further purification. The allantoic fluid of chick embryos infected with influenza H1N1 was stored and provided by Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Absorbance measurements were measured using a Nano Drop ND-1000 spectrophotometer (Thermo Fisher Scientific).
Monarch rna purification columns
Manufactured by New England Biolabs
Sourced in United States
The Monarch RNA Purification Columns are designed to efficiently extract and purify RNA from a variety of sample types. The columns utilize a silica-based membrane to bind RNA, allowing for the removal of contaminants and the subsequent elution of high-quality RNA.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rnase free water, by Sangon (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Dynabeads m 270 streptavidin, by Thermo Fisher Scientific (1 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (1 mentions)
Cleancap reagent, by TriLink (1 mentions)
2 protocols using monarch rna purification columns
1
CRISPR-Cas13a Diagnostic Assay
2
Optimized mRNA Vaccine Formulation
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mRNAs encoding for HBsAg (NCBI accession number: YP_009173871) were synthesized by T7 polymerase-mediated in vitro transcription (IVT) based on a linearized DNA template (pUC57-GW-Kan) containing codon-optimized HBsAg gene flanked with 5’ and 3’ untranslated regions (UTRs) and a 100 nt poly-A tail. During IVT procedure, mRNAs were modified with N1-Methyl-pseudouridine (Synthgene) and capped using CleanCap Reagent (TriLink). After this, IVT products were purified with Monarch RNA purification columns (NEW ENGLAND BioLabs Inc. MA, USA) and resuspended in a TE buffer at a desired concentration. For mRNA encapsulation into LNP, lipid components were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: DMG-PEG2000). The ionizable lipid (YX-02) was designed and has been patented by Firestone Biotechnologies. The lipid cocktail was mixed with mRNAs dissolved in 10 mM citrate buffer (pH4.0) at an N/P ratio of 5.3 :1 and a volume ratio of 3: 1 using a microfluidic-based equipment (INanoTML from Micro&Nano Biologics) at a total flow rate of 12 mL/min. Formulations were diluted with PBS and ultrafiltrated using 50-kDa Amicon ultracentrifugal filters. Vaccine formulation was characterized for particle diameter, polymer dispersity index (PDI) and zeta potentials using NanoBrook Omni ZetaPlus (Brookhaven Instruments).
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