Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

Rombouts, Koen; Braeckmans, Kevin; Remaut, Katrien

doi:10.1021/acs.bioconjchem.5b00579

Cited by 27 publications

(17 citation statements)

References 179 publications

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“…By counting the number of steps it is possible to calculate the amount of cargo molecules in the complex.Importantly, this method requires each NA to be labelled with exactly one fluorophore. While there are methods to do that, such as the in vitro incorporation of modified nucleotides in the polynucleotide backbone, or the introduction of specific sequences in the plasmid DNA to allow sequence-specific fluorophore attachment28 , these are rather complex procedures and need to be considered carefully. Another limitation of the method is that it is inherently limited to a low number of molecules, because else the step-wise decrease in fluorescence is buried in the overall shot-noise.…”

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confidence: 99%

Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy

et al. 2018

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Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.

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confidence: 99%

Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy

et al. 2018

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“…Also, the intensity might be dependent on environmental properties, such as the pH-dependent fluorescence of FITC fluorophores. Several fluorescent labeling strategies -with their own pros and cons -are available for NAs [52,53]. There are of course particular types of NPls, such as NPl based on AuNPs, AgNPs or TiO2 NPs, whose uptake could be determined to some extent without the need to use fluorescent labels.…”

Section: Evaluation and Quantification Of Cellular Uptake Efficiencymentioning

confidence: 99%

Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy

et al. 2018

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A plethora of biological barriers, intended to defend tissues and cells against external influences, stand in the way of efficient nucleic acid delivery by non-viral nanoplexes. Even when nanoplexes successfully evade extracellular barriers and reach their target cell, many intracellular barriers remain to be conquered. These include overcoming the plasma membrane, evading endosomal compartmentalization, and in some cases crossing the nuclear envelope. At the same time, exocytosis, autophagy and cytoplasmic degradation of the cargo should be avoided. Currently, there is a growing appreciation that the interaction of nanoplexes with these barriers should be understood in detail in order to rationally design a second generation of non-viral nanoplexes, capable of overcoming these many hurdles. Studying intracellular biobarriers is, however, quite challenging and specialized methods are constantly being developed. In this review, we present an overview of established as well as emerging techniques and assays that are currently available to the experimentalist to study nanoplexbarrier interaction, with a focus on quantitative methods.

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“…Die Fluoreszenzmarkierung von Nucleinsäuren wie DNA ist einer der einfachsten Wege zu ihrer Lokalisierung in Zellen; tatsächlich gibt es viele Methoden . Die MTase‐gesteuerte Übertragung von fluoreszierenden Gruppen bietet jedoch den besonderen Vorteil, dass die Markierung gezielt übertragen und kovalent angebracht werden kann.…”

Section: Aktuelle Und Zukünftige Anwendungenunclassified

Die Methyltransferase‐gesteuerte Markierung von Biomolekülen und ihre Anwendungen

et al. 2017

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Methyltransferasen (MTasen) bilden eine große Familie von Enzymen, die vielfältige Zielstrukturen methylieren, von DNA, RNA und Proteinen bis zu niedermolekularen Verbindungen. Die meisten MTasen nutzen S‐Adenosyl‐l‐methionin (AdoMet) als Methylquelle. In den letzten Jahren wurde intensiv an der Entwicklung von AdoMet‐Analoga gearbeitet, mit dem Ziel, andere Einheiten als einfache Methylgruppen zu übertragen. Derzeit gibt es zwei Hauptklassen von AdoMet‐Analoga – doppelt aktivierte Moleküle und Moleküle auf Aziridinbasis – die jeweils einen anderen Ansatz zur Transalkylierung des Zielmoleküls verfolgen. Dieser Aufsatz erörtert die Methoden zur Markierung und Funktionalisierung von Biomolekülen unter Verwendung von AdoMet‐abhängigen MTasen und AdoMet‐Analoga. Er behandelt die Synthesewege hin zu AdoMet‐Analoga, die Stabilität von AdoMet‐Analoga in biologischen Umgebungen und ihre Anwendung in Transalkylierungen. Schließlich werden einige Perspektiven für die Anwendung von AdoMet‐Analoga in der biologischen Forschung, Genetik oder Epigenetik und Nanotechnologie aufgezeigt.

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Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

Cited by 27 publications

References 179 publications

Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy

Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy

Methodologies to investigate intracellular barriers for nucleic acid delivery in non-viral gene therapy

Die Methyltransferase‐gesteuerte Markierung von Biomolekülen und ihre Anwendungen

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