DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

Abstract

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.

How to Tackle the Challenge of siRNA Delivery with Sequence‐Defined Oligoamino Amides

“…A step‐by‐step extension of a single siRNA revealed that a prolongation of up to 181 DNA nucleotides results in a significant improvement of transfection efficacy (Figure a). This could also be confirmed when using the cationic polymer linear PEI as a transfection agent, which is in good agreement with previous findings showing enhanced activity of linear PEI when using sticky siRNA . The stabilizing effect of the siRNA modification ideally lasts throughout the delivery process where polyplex stability is critical but should not hinder the cytosolic release of the cargo.…”

“…Another strategy to stabilize siRNA polyplexes is the conversion of single siRNA molecules into larger polyanions by hybridization, chemical ligation, click‐chemistry, and coformulation with pDNA, or other polyanions. Our group recently used DNA oligomers as adaptors to increase the size and charge of siRNA to form more stable polyplexes and thus boost transfection efficacies . Several DNA/siRNA nanostructures ranging from DNA extension of one siRNA up to structures with two to ten siRNA units were merged, and polyplexes were formed with a 3‐arm sequence‐defined oligomer.…”

“…To avoid possible steric hindrance of the interaction of siRNA with the RISC, a bioreducible disulfide linker between the siRNA passenger strand and the DNA extension was introduced. In this way, the siRNA is liberated from the DNA adaptor in the cytosol (Figure b).…”

“…b) Gene silencing of folic acid targeted polymer 356 with bioreducibly extended siRNA and bioreducibly attached lytic peptide INF7. Reproduced with permission . Copyright 2016, Macmillan Publishers Ltd.…”

“…The Inf7 peptide can be attached directly to the nucleic acid to improve the delivery of siRNA. Both the bioreversible attachment of Inf7 to the 5'‐end of siRNA with a C6‐ss‐C6 spacer and linkage to the 5′ DNA adaptor of DNA‐extended siRNA (Figure b) lead to significantly enhanced gene knockdown of PEGylated FolA‐targeted polyplexes. Inf7 was also an indispensable element of post‐modified TfR targeted siRNA polyplexes .…”