Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2023 May 11;14:2657. doi: 10.1038/s41467-023-38244-8

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 5 — a–f RP-HPLC analysis of IVT-RNAs. PureCap analogs TriPure_0 (a), TriPure_1 (b), TetraPure_2 (c), and TeraPure_2/m6A (d) were added to the reaction. Control mRNA using cap analogs Tri_1 (e) or Tetra_2 (f) was transcribed from a DNA template containing the A-inserted promoter. The transcript was analyzed as a crude mixture (i, black line) or after being purified by preparative HPLC (ii, blue line). Purified RNA was analyzed after deprotection by irradiating 365-nm light (iii, green line). The elution time (min) of the peak was noted nearby. A ratio (%) of capped mRNA calculated based on the peak area was listed in parentheses after the elution time. g‒i Analysis of purified mRNA used to measure the biological activities. The mRNAs were named after the cap analog used. Cap(−) means no cap analog was added. AnP means it was further treated with Antarctic phosphatase. g dPAGE analysis of the 5′ end of purified mRNA cleaved with DNAzyme 10–23 to assess its capping state. The capped ratio was calculated based on the band intensities of the capped and uncapped RNA fragments. h dPAGE analysis of purified mRNAs (25 ng each). Lane 1, Cap(−); 2, TriPure_0; 3, TriPure_1; 4, TetraPure_2; 5, TetraPure_2/m6A; 6, Tri_1; 7, Tri_1/AnP; 8, Tetra_2; 9, Tetra_2/AnP. h Removal of dsRNA from purified mRNA was confirmed by dot-blot assay using anti-dsRNA J2 antibody. 250 ng mRNAs were spotted. As a positive control of the assay, the dsRNA sample was dotted in spots 1 to 5 (0, 0.050, 0.50, 5.0, and 50 ng, respectively). Analyzed mRNAs were as follows; spot 6, mRNA before HPLC purification prepared with no cap analog; 7, Cap(−); 8, TriPure_0; 9, TriPure_1; 10, TetraPure_2; 11, TetraPure_2/m6A; 12, Tri_1; 13, Tri_1/AnP; 14, Tetra_2; 15, Tetra_2/AnP. a‒i Each experiment was repeated independently at least three times to obtain similar results. Source Data were provided with this paper.