Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes

Troiber, Christina; Edinger, Daniel; Kos, Petra; Schreiner, L; Kläger, Raphaela; Herrmann, Annika; Wagner, Ernst

doi:10.1016/j.biomaterials.2012.11.021

Cited by 90 publications

(174 citation statements)

References 31 publications

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“…The fatty acid unit is connected to two identical arms consisting of two Stp units, three tyrosines for stabilization, and a terminal cysteine ( Supplementary Figure S9 ). 39 …”

Section: Resultsmentioning

confidence: 99%

DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

Heissig

Klein

Hadwiger³

et al. 2016

Molecular Therapy - Nucleic Acids

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siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4–10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3′ end of the siRNA passenger strand was beneficial over the 5′ end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol–shielded polyplex to downregulate a luciferase gene in folate receptor–positive cells.

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“…The fatty acid unit is connected to two identical arms consisting of two Stp units, three tyrosines for stabilization, and a terminal cysteine ( Supplementary Figure S9 ). 39 …”

Section: Resultsmentioning

confidence: 99%

DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

Heissig

Klein

Hadwiger³

et al. 2016

Molecular Therapy - Nucleic Acids

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“…These oligomers provide excellent nucleic acid binding ability, endosomal buffering capacity, and site-specific positioning of multiple functionalities. Further studies have been undertaken to optimize such cationic carriers by systematic variation of the topology [31], inclusion of small chemical delivery motifs such as buffering histidines [32], fatty acids [33], tyrosine trimers [10], disulfide-forming units [34], targeting ligands [35][36][37][38][39][40][41][42] and combinations of such elements [43]. Accordingly, this strategy of a step-by-step optimization resulted in a library of >1000 oligomers which included precise siRNA carriers.…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, great efforts have been made in the development of various siRNA carriers. Polymeric materials [5][6][7][8][9][10][11][12][13][14][15][16][17], liposomes [18,19], inorganic platforms [20] and hybrid systems [21][22][23][24] all showed their potential [25]. However, systemic delivery of siRNA directed to the tumor site remains a major limitation.…”

Section: Introductionmentioning

confidence: 99%

Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes

Lee

Kessel

et al. 2016

Journal of Controlled Release

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“…The sequence-defined lipo-oligomer 278 was synthesized by solid-phase assisted synthesis as described in our previous publications [11, 29, 31]. It is a U-shaped lipo-oligocation consisting of a protonable backbone of three succinoyl -tetraethylene pentamine (Stp) units for complexation of the nucleic acids.…”

Section: Purification With High-performance Liquid Chromatographymentioning

confidence: 99%

Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

Heissig

Schrimpf

Hadwiger³

et al. 2017

PLoS ONE

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Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.

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Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes

Cited by 90 publications

References 31 publications

DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes

Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

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