In Vitro Transcription of Modified mRNAs

Chemically modified, co-transcriptionally capped Cap 1 Cas9 and firefly luciferase (FLuc) mRNAs were synthesized by T7 RNA polymerase in vitro transcription. All enzymes were purchased from New England Biolabs (Ipswich, MA). Transcriptions were done in 1× transcription buffer (40 mM Tris, 10 mM dithiothreitol, 2 mM spermidine, 0.002% Triton X-100, and 27 mM magnesium acetate) using final concentrations of 8 U/μL T7 RNA polymerase (M0251L); 0.002 U/μL inorganic pyrophosphatase (M2403L); 1 U/μL murine RNase inhibitor (M0314L); 0.025 μg/μL standard or uridine-depleted transcription template; 5 mM CleanCap Cap 1 AG trimer; and 5 mM each of ATP, cytidine triphosphate (CTP) (or CTP analog), GTP, and uridine triphosphate (UTP) (or UTP analog), as indicated in Table 1. Transcription reactions were incubated at 37°C for 2 hr and treated with final 0.4 U/μL DNase I (M0303L) in 1× DNase I buffer for 15 min at 37°C. We initially made anti-reverse CleanCap trimers with a 3′-O-methyl group on the sugar of the ^m7G to prevent incorporation in the opposite orientation, but we found this to be unnecessary, as the 3′-O-methyl version functioned equivalently to CleanCap with a 3′ OH. mRNAs were purified by RNeasy Maxi (QIAGEN, 75162), phosphatase treated for 1 hr with final 0.25 U/μg Antarctic phosphatase (M0289L) in 1× Antarctic phosphatase buffer, and then re-purified by RNeasy. A portion of each mRNA was purified by HPLC as described by Kariko et al.,60 except that mRNA was recovered from HPLC fractions by RNeasy purification. Purification was carried out on a PRP-H1 column (Hamilton Company) at 65°C using a gradient of 100 mM triethylammonium acetate/acetonitrile. Transcription quality was measured by bioanalyzer analysis (Agilent 2100 Bioanalyzer). mRNA concentrations were measured by UV spectroscopy and corrected for predicted extinction coefficient.