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Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts

Heyang Zhang (UGent) , Koen Rombouts (UGent) , Laurens Raes (UGent) , Ranhua Xiong (UGent) , Stefaan De Smedt (UGent) , Kevin Braeckmans (UGent) and Katrien Remaut (UGent)
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Abstract
Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is approximate to 1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics.
Keywords
extracellular degradation, fluorescence correlation spectroscopy, intracellular degradation, nucleic acids, single particle tracking, SINGLE-PARTICLE TRACKING, SODIUM DODECYL-SULFATE, NUCLEIC-ACID DELIVERY, PHOSPHODIESTER OLIGONUCLEOTIDES, CORRELATION SPECTROSCOPY, GENE-TRANSFER, IN-VITRO, DEGRADATION, TRANSFECTION, RIBONUCLEASE

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Citation

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MLA
Zhang, Heyang, et al. “Fluorescence-Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts.” ADVANCED BIOSYSTEMS, vol. 4, no. 5, 2020, doi:10.1002/adbi.202000057.
APA
Zhang, H., Rombouts, K., Raes, L., Xiong, R., De Smedt, S., Braeckmans, K., & Remaut, K. (2020). Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts. ADVANCED BIOSYSTEMS, 4(5). https://doi.org/10.1002/adbi.202000057
Chicago author-date
Zhang, Heyang, Koen Rombouts, Laurens Raes, Ranhua Xiong, Stefaan De Smedt, Kevin Braeckmans, and Katrien Remaut. 2020. “Fluorescence-Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts.” ADVANCED BIOSYSTEMS 4 (5). https://doi.org/10.1002/adbi.202000057.
Chicago author-date (all authors)
Zhang, Heyang, Koen Rombouts, Laurens Raes, Ranhua Xiong, Stefaan De Smedt, Kevin Braeckmans, and Katrien Remaut. 2020. “Fluorescence-Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts.” ADVANCED BIOSYSTEMS 4 (5). doi:10.1002/adbi.202000057.
Vancouver
1.
Zhang H, Rombouts K, Raes L, Xiong R, De Smedt S, Braeckmans K, et al. Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts. ADVANCED BIOSYSTEMS. 2020;4(5).
IEEE
[1]
H. Zhang et al., “Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts,” ADVANCED BIOSYSTEMS, vol. 4, no. 5, 2020.
@article{8659890,
  abstract     = {Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is approximate to 1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics.},
  articleno    = {2000057},
  author       = {Zhang, Heyang and Rombouts, Koen and Raes, Laurens and Xiong, Ranhua and De Smedt, Stefaan and Braeckmans, Kevin and Remaut, Katrien},
  issn         = {2366-7478},
  journal      = {ADVANCED BIOSYSTEMS},
  keywords     = {extracellular degradation,fluorescence correlation spectroscopy,intracellular degradation,nucleic acids,single particle tracking,SINGLE-PARTICLE TRACKING,SODIUM DODECYL-SULFATE,NUCLEIC-ACID DELIVERY,PHOSPHODIESTER OLIGONUCLEOTIDES,CORRELATION SPECTROSCOPY,GENE-TRANSFER,IN-VITRO,DEGRADATION,TRANSFECTION,RIBONUCLEASE},
  language     = {eng},
  number       = {5},
  pages        = {16},
  title        = {Fluorescence-based quantification of messenger RNA and plasmid DNA decay kinetics in extracellular biological fluids and cell extracts},
  url          = {http://dx.doi.org/10.1002/adbi.202000057},
  volume       = {4},
  year         = {2020},
}

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