Analysis and purification of ssRNA and dsRNA molecules using asymmetrical flow field flow fractionation

Eskelin, Katri; Lampi, Mirka; Coustau, Christine; Imani, Jafargholi; Kögel, Karl-Heinz; M, Poranen Minna

doi:10.1016/j.chroma.2022.463525

Cited by 9 publications

(5 citation statements)

References 54 publications

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“…Using fluorescence correlation spectroscopy, Borodavka et al (Borodavka et al 2016 ) have found the hydrodynamic radius of 960-, 1200-, 1400-, and 1800 nt-long RNA molecules to be 8.8–11.9 nm. This concurs with AF4 (asymmetrical flow field-flow fractionation) measurements on single-stranded RNA molecules of 500–1800 nt length with hydrodynamic radii ranging from 8.4 to 14.7 nm (Eskelin et al 2022 ). Considering the hydrodynamic radius of mRNAs and our LNP preparation (25–100 nm, Fig.…”

Section: Resultssupporting

confidence: 88%

Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation

Bepperling

Richter

2023

Eur Biophys J

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Lipid nanoparticles as delivery system for mRNA have recently attracted attention to a broader audience as COVID-19 mRNA vaccines. Their low immunogenicity and capability to deliver a variety of nucleic acids renders them an interesting and complementary alternative to gene therapy vectors like AAVs. An important quality attribute of LNPs is the copy number of the encapsulated cargo molecule. This work describes how density and molecular weight distributions obtained by density contrast sedimentation velocity can be used to calculate the mRNA copy number of a degradable lipid nanoparticle formulation. The determined average copy number of 5 mRNA molecules per LNP is consistent with the previous studies using other biophysical techniques, such as single particle imaging microscopy and multi-laser cylindrical illumination confocal spectroscopy (CICS). Supplementary Information The online version contains supplementary material available at 10.1007/s00249-023-01663-y.

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Section: Resultssupporting

confidence: 88%

Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation

Bepperling

Richter

2023

Eur Biophys J

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“…In order to define if the protection provided by Pmk1-dsRNA against Mo infection (Fig. 8) was mediated by RNAi pathway or PTI, we performed an experiment in which we treated plants with Mo-specific and nonspecific dsRNA and inoculated dsRNA-treated plants with Mo after 7 or 14 d. As non-targeting dsRNA, we used bacteriophage phi6-specific dsRNA, which can be produced in high quantities using a cell-based production system we have developed earlier 35,36 .…”

Section: Dsrna Induced Rnai Protects Plants Against Magnaporthe Oryza...mentioning

confidence: 99%

Exogenous dsRNA-Mediated Control of Rice Blast Fungus Involves Sequence-Specific RNAi and Sequence-Unspecific Fungal Stress Responses

Ladera-Carmona,

Zheng,

Moorlach

et al. 2024

Preprint

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In vertebrates and plants, double-stranded (ds)RNA plays crucial roles as a pathogen-associated molecular pattern (PAMP) and as a mediator of RNA interference (RNAi). However, our understanding of fungal responses to dsRNA remains limited. Here, we demonstrate that Magnaporthe oryzae (Mo), a globally significant crop pathogen, actively internalizes exogenous dsRNA across a broad size range of 21 to about 3000 bp. Treatment of Mo conidia with dsRNA, irrespective of size or sequence, induces aberrant germ tube elongation. Additionally, application of dsRNA to Brachypodium distachyon leaves suppresses disease progression upon Mo infection. Intriguingly, dsRNA elicits canonical stress pathways in Mo, as evidenced by nuclear accumulation of the stress marker mitogen-activated protein kinase Hog1p and the production of reactive oxygen species. However, these sequence-nonspecific effects are transient, while sequence-specific RNAi activity predominates in a preventive intervention scenario, when a time interval of several days between dsRNA application and fungal inoculation is maintained, and dsRNA is stabilized in novel alginate-chitosan nanoparticles. Overall, our data show that the effects of dsRNA on fungi and the diseases they cause can be multifaceted and complex, which has implications for the development and application of dsRNA pesticides in agricultural practice.

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“…Although the exact source of this response is not yet fully elucidated, it is likely that residual dsRNA species are still present in highly purified mRNA. Many attempts are made to both identify this contaminant and to find RNAPs that result in lower production of dsRNA [ 2 , 4 , 5 , 11 ]. Examples of the latter include rationally engineered T7 RNAP [ 12 ], thermostable T7 RNAP [ 5 ], and the psychrophilic phage VSW3 RNAP [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…This antibody, however, cannot detect dsRNA shorter than 43 bp, and it is unclear to what extent the J2 mAb binds ssRNA secondary structures [ 14–16 ]. Analytical methods to detect dsRNA include HPLC, mass spectrometry (MS), and asymmetric field flow fractionation (AF4) [ 11 , 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts

Francis,

Frida J,

Thanh-Huong

et al. 2024

RNA Biology

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The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

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Analysis and purification of ssRNA and dsRNA molecules using asymmetrical flow field flow fractionation

Cited by 9 publications

References 54 publications

Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation

Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation

Exogenous dsRNA-Mediated Control of Rice Blast Fungus Involves Sequence-Specific RNAi and Sequence-Unspecific Fungal Stress Responses

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts

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