In silico study of PEI-PEG-squalene-dsDNA polyplex formation: the delicate role of the PEG length in the binding of PEI to DNA

Using a two step simulation protocol the atomistic interactions between PEG and b-PEI and the effect of these interactions on DNA binding were determined

Anwendung eines neuen Fluoreszenz-Resonanz-Energie-Transfer (FRET) Systems in synthetischer DNA

Die vorgelegte Arbeit beschreibt die Anwendung von neuen, robusten und sehr empfindlichen FRET Systemen in DNA. Die untersuchten Systeme basieren auf Ru(II)-bathophenanthrolin Komplexen als FRET-Akzeptoren und einem Quinolinon Chromophor als FRET-Donor. Der Ru(II)-bathophenanthrolin Komplex konnte in einfachen Schritten in hohen Ausbeuten synthetisiert und gereinigt werden, um ihn anschließend an entsprechende aminomodifizierte Oligonucleotide koppeln zu können. Für die kovalente Fixierung des FRET-Donors an Oligonucleotide via einer post-synthetischen Strategie, wurde das Carbostyril Derivat in eine Carbonsäure überführt, die als N-hydroxysuccinimid-Ester aktiviert wurde. Absorptions- und Emissionsspektren verschiedener Oligonucleotide, welche entweder den Donor- oder den Akzeptor Farbstoff enthielten, zeigten eine gute Spektrale Überlappung der Emissions-Bande des Donors mit der Absorptions-Bande des Akzeptors, eine entscheidende Vorraussetzung für einen starken FRET. In der ersten Phase der Dissertation lag der Schwerpunkt auf der Synthese von DNA Strängen, welche sowohl mit dem Donor als auch dem Akzeptor über einen post synthetischen Ansatz markiert wurden. Der FRET-Donor wurde dabei an die Aminofunktion einer 5-(3-Aminoprop-1-ynyl)-2´-desoxyuridin Einheit im synthetisierten Oligonukleotid über eine Amidbindung gekuppelt. Das 5´-Ende des Oligonucleotids, welches ein modifiziertes 5´-Amino-5‘-desoxy Motif enthielt, wurde danach mit dem aktivierten FRET-Akzeptor gekuppelt. Bei einer verbesserten Markierungsmethode lag das Augenmerk auf einer pre-synthetitischen Strategie, d.h., Einbau des Chromophors in das Oligonucleotid während seiner Synthese an der festen Phase. Dies erforderte die Synthese von zwei speziell modifizierten Bausteinen, die den Donor direkt an die Base, in diesem Fall Uracil bzw. Adenin, gebunden haben. Diese Phosphoramidite liessen sich mit hoher Effizienz während der Festphasensynthese einbauen. Der FRET wurde dann in Einzelstrang DNA untersucht, die Donor, Akzeptor und einen Uridin Rest als Sollbruchstelle zur Spaltung im basischen Medium enthielt. Im ungespalten Oligomeren wurde ein starker FRET beobachtet, der nach der Spaltung nicht mehr zu beobachten war. Alternativ wurde auch noch eine sauer spaltbare Phosphamidbindung als Spaltstelle eingebaut. Aufgrund der langen Abklingzeit für die Fluoreszenz konnten auch zeitaufgelöste Messungen durchgeführt werden.The presented work describes the application of new, robust and highly sensitive FRET systems in DNA. The investigated systems are based on a Ru(II) -bathophenanthroline complex as FRET-acceptor and quinolinone derivative as FRET-donor. The Ru (II)-bathophenanthroline complex was synthesized by a straightforward route in high yield for the coupling to corresponding amino-modified oligonucleotides. For the covalent attachment of the FRET-donor to oligonucleotides via a post-synthetic strategy, the carboxyl function of the carbostyril derivative was activated as N-hydroxysuccinimid-ester. The absorption and emission spectra of separate oligonucleotides bearing either only the donor or the acceptor chromophore showed a good spectral overlap, a crucial requirement for a strong FRET. The first part of the current thesis was focused on the labelling of DNA with both chromophores by a post synthetic approach. The FRET-donor was attached to a 5-(3-aminoprop-1-ynyl)-2’-deoxyuridine unit via an amide bond. The 5’-end of the oligonucleotides consisting of a modified 5’-amino-5’-deoxy unit was then coupled to the activated FRET-acceptor. An improved labelling method focussed on a pre-synthetic strategy, i.e., incorporation of the chromophore molecules into oligonucleotides during their chemical synthesis on solid support. This required the synthesis of two special building blocks in which the donor chromophore was attached to uracil or adenin, respectively. Both phosphoramidites could be introduced with high efficiency during solid phase synthesis. Since it was also possible to incorporate the Ru-complex during the solid phase approach, the whole procedure for the incorporation of the FRET into synthetic DNA is straightforward and very efficient. The FRET was investigated in single stranded DNA bearing donor, acceptor and an uridine moiety as specific cleavage site. In the intact fragment a high FRET could be observed which diminished after cleavage under basic conditions. As an alternative potential cleavage site to be broken under acidic conditions, we have inserted a phosphamide bond. Due to the long decay time of the fluorescence of the Ru complex, time-resolved measurements could also be carried out successfully

Tunable Composition of Dynamic Non-Viral Vectors over the DNA Polyplex Formation and Nucleic Acid Transfection

Polyethylene glycol (PEG) functionalization of non-viral vectors represents a powerful tool through the formation of an overall surface charge shielding ability, which is fundamental for efficient nucleic acid delivery systems. The degree of non-viral vector PEGylation and the molecular weight of utilized PEG is crucial since the excessive use of PEG units may lead to a considerable reduction of the DNA-binding capacity and, subsequently, in a reduction of in vitro transfection efficiency. Herein, we report a detailed study on a series of dynamic combinatorial frameworks (DCFs) containing PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) of different lengths, and branched low molecular weight polyethylenimine components, reversibly connected in hyperbranched structures, as efficient dynamic non-viral vectors. The obtained frameworks were capable of forming distinct supramolecular amphiphilic architectures, shown by transmission electron microscopy (TEM) and dynamic light scattering (DLS), with sizes and stability depending on the length of PEG units. The interaction of PEGylated DCFs with nucleic acids was investigated by agarose gel retardation assay and atomic force microscopy (AFM), while their transfection efficiency (using pCS2+MT-Luc DNA as a reporter gene) and cytotoxicity were evaluated in HeLa cells. In addition, the data on the influence of the poly-(ethyleneglycol)-bis(3-aminopropyl) length in composition of designed frameworks over transfection efficiency and tolerance in human cells were analyzed and compared

DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection

International audienc

Optimization of Polyplex Formation between DNA Oligonucleotide and Poly(ʟ-Lysine): Experimental Study and Modeling Approach

The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(ʟ-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4

Experimental design, modeling and optimization of polyplex formation between DNA oligonucleotides and branched polyethylenimine

International audienc

Synergistic Effect of Low Molecular Weight Polyethylenimine and Polyethylene Glycol Components in Dynamic Nonviral Vector Structure, Toxicity, and Transfection Efficiency

When studying polyethylenimine derivatives as nonviral vectors for gene delivery, among the important issues to be addressed are high toxicity, low transfection efficiency, and nucleic acid polyplex condensation. The molecular weight of polyethylenimine, PEGylation, biocompatibility and, also, supramolecular structure of potential carrier can all influence the nucleic acid condensation behavior, polyplex size, and transfection efficiency. The main challenge in building an efficient carrier is to find a correlation between the constituent components, as well as the synergy between them, to transport and to release, in a specific manner, different molecules of interest. In the present study, we investigated the synergy between components in dynamic combinatorial frameworks formed by connecting PEGylated squalene, poly-(ethyleneglycol)-bis(3-aminopropyl) and low molecular weight polyethylenimine components to 1,3,5-benzenetrialdehyde, via reversible imine bond, applying a dynamic combinatorial chemistry approach. We report comparative structural and morphological data, DNA binding affinity, toxicity and transfection efficiency concerning the ratio of polyethylenimine and presence or absence of poly-(ethyleneglycol)-bis(3-aminopropyl) in composition of dynamic combinatorial frameworks. In vitro biological assessments have revealed the fact that nonviral vectors containing poly-(ethyleneglycol)-bis(3-aminopropyl) and the lowest amount of polyethylenimine have significant transfection efficiency at N/P 50 ratio and display insignificant cytotoxicity on the HeLa cell line