从体外转录mRNA中选择性去除dsRNA的氧化石墨烯修饰树脂。

IF 4.7 Q2 MATERIALS SCIENCE, BIOMATERIALS
ACS Applied Bio Materials Pub Date : 2025-04-21 Epub Date: 2025-03-27 DOI:10.1021/acsabm.5c00320
Junhyung Ryu, Jayoung Namgung, Jinmin Jang, Goeun Lee, Kwanghee Yoo, Bong-Hyun Jun, Dong-Eun Kim
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引用次数: 0

摘要

信使RNA (mRNA)已被证明是对抗意外大流行的有效疫苗剂,具有快速生产针对特定病原体的定制治疗药物的优势。然而,在体外转录(IVT)反应中产生的不希望的副产物,如双链RNA (dsRNA),可能会阻碍翻译效率,并在mRNA摄取后触发细胞中的炎症细胞因子。在这项研究中,我们开发了一种简单的方法,使用聚乙二醇化聚苯乙烯树脂,用氧化石墨烯(GO@PEG-PS)进一步表面修饰,从IVT mRNA中去除dsRNA。由于氧化石墨烯(GO)的特性,GO@PEG-PS树脂吸附mRNA,在Mg2+存在下,它优先吸附单链核酸而不是双链核酸。树脂结合的单链RNA很容易被EDTA和尿素的混合物解吸,可能分别通过螯合Mg2+和破坏氢键来解吸。使用GO@PEG-PS对IVT mRNA进行自旋柱层析,消除了至少80%的dsRNA,回收了大约85%的mRNA。此外,该程序排除了IVT反应后的盐沉淀步骤,该步骤从IVT组分(包括核苷酸和酶)中分离mrna。纯化后的mRNA转染后,蛋白翻译增强,干扰素(IFN)-β分泌减少。我们预计,采用GO@PEG-PS树脂的mRNA纯化色谱系统将有助于去除mRNA生产过程中的dsRNA污染。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Graphene Oxide-Modified Resin for Selective dsRNA Removal from In Vitro-Transcribed mRNA.

Messenger RNA (mRNA) has proven to be an effective vaccine agent against unexpected pandemics, offering the advantage of rapidly producing customized therapeutics targeting specific pathogens. However, undesired byproducts, such as double-stranded RNA (dsRNA), generated during in vitro transcription (IVT) reactions may impede translation efficiency and trigger inflammatory cytokines in cells after mRNA uptake. In this study, we developed a facile method using PEGylated polystyrene resins that were further surface-modified with graphene oxide (GO@PEG-PS) for the removal of dsRNA from IVT mRNA. The GO@PEG-PS resin adsorbed mRNA due to the property of graphene oxide (GO), which preferentially adsorbs single-stranded nucleic acids over double-stranded nucleic acids in the presence of Mg2+. The resin-bound single-stranded (ss) RNA was readily desorbed with a mixture of EDTA and urea, possibly by chelating Mg2+ and disrupting hydrogen bonding, respectively. Spin-column chromatography with GO@PEG-PS for IVT mRNA eliminated at least 80% of dsRNA, recovering approximately 85% of mRNA. Furthermore, this procedure precluded the salt precipitation step after the IVT reaction, which fractionates mRNAs from the IVT components, including nucleotides and enzymes. The purified mRNA exhibited enhanced protein translation with reduced secretion of interferon (IFN)-β upon mRNA transfection. We anticipate that the mRNA purification chromatography system employing GO@PEG-PS resin will facilitate the removal of dsRNA contamination during mRNA production.

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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
期刊介绍: ACS Applied Bio Materials is an interdisciplinary journal publishing original research covering all aspects of biomaterials and biointerfaces including and beyond the traditional biosensing, biomedical and therapeutic applications. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrates knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important bio applications. The journal is specifically interested in work that addresses the relationship between structure and function and assesses the stability and degradation of materials under relevant environmental and biological conditions.
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