Related Experiment Video
Updated: Jan 31, 2026
11:22
Measuring the Kinetics of mRNA Transcription in Single Living Cells
Published on: August 25, 2011
16.1K
ShortCut RNase III is capable of digesting single mRNA transcripts into < 10 nucleotide products
Kyle J Tynan1, Mark S Lowenthal2, Karndeep Singh3
1National Institute of Standards and Technology (NIST), 100 Bureau Dr, Gaithersburg, MD 20899 USA; Institute for Bioscience and Biotechnology Research (IBBR), 9600 Gudelsky Dr, Rockville, MD 20850 USA.
Biochimie
|January 29, 2026
Summary
ShortCut RNase III unexpectedly degrades single-stranded Cas9 mRNA into small fragments. This enzyme
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Therapeutics
Background:
- RNase III is a double-stranded RNA (dsRNA) specific ribonuclease.
- It is commonly used to remove dsRNA impurities from in vitro transcription (IVT) products.
- Ensuring mRNA purity is critical for therapeutic applications.
Purpose of the Study:
- To investigate the substrate specificity of ShortCut RNase III.
- To evaluate its efficacy in degrading dsRNA impurities in mRNA manufacturing.
- To assess potential unintended degradation of single-stranded mRNA.
Main Methods:
- Utilized ShortCut RNase III, a recombinant E. coli RNase III fusion protein.
- Assessed enzymatic activity on both dsRNA and single-stranded Cas9 mRNA.
- Analyzed degradation products using nucleotide fragment length analysis.
Main Results:
- ShortCut RNase III degraded dsRNA to 18-25 base pair fragments as expected.
- Unexpectedly, the enzyme also degraded single-stranded Cas9 mRNA.
- Degradation of single-stranded mRNA resulted in fragments as short as 9 nucleotides.
Conclusions:
- ShortCut RNase III exhibits broader substrate specificity than previously reported.
- Its activity extends to single-stranded mRNA, posing risks for mRNA manufacturing.
- Further evaluation is needed to determine its suitability for dsRNA removal in therapeutic mRNA production.
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