Dynamic Nanostructures for Conditional Activation and Deactivation of Biological Pathways

Abstract

Nucleic acid nanotechnology utilizes natural and synthetic structural motifs to build versatile nucleic acid nanoparticles (NANPs). These rationally designed assemblies can be further equipped with functional nucleic acids and other molecules such as peptides, fluorescent dyes, etc. In addition to nucleic acids that directly interact with the regulated target gene transcripts, NANPs can display decoys, wherein the oligonucleotide stretches with transcription factor binding sequences, preventing transcription initiation. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a group of five crucial transcription factors regulating the pathogenesis of inflammatory diseases and cancer; as such, they are relevant targets for therapy. One therapeutic approach involves interdependent self-recognizing hybridized DNA/RNA fibers designed to bind NF-κB and prevent its interaction with the promotor region of NF-κB-dependent genes involved in inflammatory responses. Decoying NF-κB results in the inability to initiate transcription of regulated genes, showing a promising approach to gene regulation and gene therapy. The protocol described herein provides detailed steps for the synthesis of NF-κB decoy fibers, as well as their characterization using polyacrylamide gel electrophoresis (to confirm desired physicochemical properties and purity) and functional bioassays (to confirm desired biological activity).

Keywords: Decoys; Hybrid fibers; NF-κB regulation; Nucleic acid nanoparticles (NANPs).