An immuno-northern technique to measure the size of dsRNA byproducts in in vitro transcribed RNA

IF 3 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

ELECTROPHORESIS Pub Date : 2024-05-24 DOI:10.1002/elps.202400036

Nathaniel E. Clark, Matthew R. Schraut, Roger A. Winters, Kelley Kearns, Thomas C. Scanlon

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Abstract

Double-stranded RNA is an immunogenic byproduct present in RNA synthesized with in vitro transcription. dsRNA byproducts engage virus-sensing innate immunity receptors and cause inflammation. Removing dsRNA from in vitro transcribed messenger RNA (mRNA) reduces immunogenicity and improves protein translation. Levels of dsRNA are typically 0.1%–0.5% of total transcribed RNA. Because they form such a minor fraction of the total RNA in transcription reactions, it is difficult to confidently identify discrete bands on agarose gels that correspond to the dsRNA byproducts. Thus, the sizes of dsRNA byproducts are largely unknown. Total levels of dsRNA are typically assayed with dsRNA-specific antibodies in ELISA and immuno dot-blot assays. Here we report a dsRNA-specific immuno-northern blot technique that provides a clear picture of the dsRNA size distributions in transcribed RNA. This technique could complement existing dsRNA analytical methods in studies of dsRNA byproduct synthesis, dsRNA removal, and characterization of therapeutic RNA drug substances.

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一种用于测量体外转录 RNA 中 dsRNA 副产物大小的免疫北方技术。

双链 RNA 是体外转录合成的 RNA 中的一种免疫原性副产物。从体外转录的信使 RNA（mRNA）中去除 dsRNA 可降低免疫原性并改善蛋白质翻译。dsRNA通常占转录 RNA 总量的 0.1%-0.5%。由于dsRNA 在转录反应中只占总 RNA 的很小一部分，因此很难在琼脂糖凝胶上确定与 dsRNA 副产物相对应的离散条带。因此，dsRNA 副产物的大小在很大程度上是未知的。在酶联免疫吸附和免疫点印迹分析中，dsRNA 的总水平通常是用 dsRNA 特异性抗体来检测的。在这里，我们报告了一种dsRNA特异性免疫北方印迹技术，它能清楚地显示转录 RNA 中 dsRNA 的大小分布。这项技术可以补充现有的dsRNA分析方法，用于研究dsRNA副产物的合成、dsRNA的去除以及治疗性RNA药物物质的表征。

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来源期刊

ELECTROPHORESIS 生物-分析化学

CiteScore

6.30

自引率

13.80%

发文量

244

审稿时长

1.9 months

期刊介绍： ELECTROPHORESIS is an international journal that publishes original manuscripts on all aspects of electrophoresis, and liquid phase separations (e.g., HPLC, micro- and nano-LC, UHPLC, micro- and nano-fluidics, liquid-phase micro-extractions, etc.). Topics include new or improved analytical and preparative methods, sample preparation, development of theory, and innovative applications of electrophoretic and liquid phase separations methods in the study of nucleic acids, proteins, carbohydrates natural products, pharmaceuticals, food analysis, environmental species and other compounds of importance to the life sciences. Papers in the areas of microfluidics and proteomics, which are not limited to electrophoresis-based methods, will also be accepted for publication. Contributions focused on hyphenated and omics techniques are also of interest. Proteomics is within the scope, if related to its fundamentals and new technical approaches. Proteomics applications are only considered in particular cases. Papers describing the application of standard electrophoretic methods will not be considered. Papers on nanoanalysis intended for publication in ELECTROPHORESIS should focus on one or more of the following topics: • Nanoscale electrokinetics and phenomena related to electric double layer and/or confinement in nano-sized geometry • Single cell and subcellular analysis • Nanosensors and ultrasensitive detection aspects (e.g., involving quantum dots, "nanoelectrodes" or nanospray MS) • Nanoscale/nanopore DNA sequencing (next generation sequencing) • Micro- and nanoscale sample preparation • Nanoparticles and cells analyses by dielectrophoresis • Separation-based analysis using nanoparticles, nanotubes and nanowires.