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Fluorescence Correlation Spectroscopy to find the critical balance between extracellular association and intracellular dissociation of mRNA complexes
- Source :
- ACTA BIOMATERIALIA
- Publication Year :
- 2018
- Publisher :
- Elsevier BV, 2018.
-
Abstract
- Fluorescence Correlation Spectroscopy (FCS) is a promising tool to study interactions on a single molecule level. The diffusion of fluorescent molecules in and out of the excitation volume of a confocal microscope leads to the fluorescence fluctuations that give information on the average number of fluorescent molecules present in the excitation volume and their diffusion coefficients. In this context, we complexed mRNA into lipoplexes and polyplexes and explored the association/dissociation degree of complexes by using gel electrophoresis and FCS. FCS enabled us to measure the association and dissociation degree of mRNA-based complexes both in buffer and protein-rich biological fluids such as human serum and ascitic fluid, which is a clear advantage over gel electrophoresis that was only applicable in protein-free buffer solutions. Furthermore, following the complex stability in buffer and biological fluids by FCS assisted to understand how complex characteristics, such as charge ratio and strength of mRNA binding, correlated to the transfection efficiency. We found that linear polyethyleneimine prevented efficient translation of mRNA, most likely due to a too strong mRNA binding, whereas the lipid based carrier Lipofectamine® messengerMAX did succeed in efficient release and subsequent translation of mRNA in the cytoplasm of the cells. Overall, FCS is a reliable tool for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production. Statement of Significance The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, …), but should release the mRNA again in the intracellular environment. In this paper, we evaluated a method (Fluorescence Correlation Spectroscopy) that allows for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production.
- Subjects :
- 0301 basic medicine
NUCLEIC-ACID DELIVERY
Biomedical Engineering
Fluorescence correlation spectroscopy
02 engineering and technology
OLIGONUCLEOTIDE COMPLEXES
Biochemistry
GENE DELIVERY
TRANSGENE EXPRESSION
Biomaterials
03 medical and health sciences
mRNA complex formation
POLYETHYLENIMINE
Cell Line, Tumor
mRNA delivery
Biofluids
Medicine and Health Sciences
Protein biosynthesis
Extracellular
Humans
DNA DELIVERY
RNA, Messenger
Molecular Biology
IN-VIVO
Gel electrophoresis
Messenger RNA
STABILITY
Chemistry
TRANSFECTION EFFICIENCY
Biology and Life Sciences
FCS
General Medicine
Transfection
021001 nanoscience & nanotechnology
Spectrometry, Fluorescence
030104 developmental biology
Cytoplasm
Biophysics
0210 nano-technology
Cell-Free Nucleic Acids
STEM-CELLS
Complex stability
Intracellular
Biotechnology
Subjects
Details
- ISSN :
- 17427061
- Volume :
- 75
- Database :
- OpenAIRE
- Journal :
- Acta Biomaterialia
- Accession number :
- edsair.doi.dedup.....b56932a928deba56985df504548dd4fb