Context-Sensitive Cleavage of Folded DNAs by Loop-Targeting bPNAs.

Herein, we demonstrate context-dependent molecular recognition of DNA by synthetic bPNA iron and copper complexes, using oxidative backbone cleavage as a chemical readout for binding. Oligoethylenimine bPNAs displaying iron·EDTA or copper·phenanthroline sites were found to be efficient chemical nucleases for designed and native structured DNAs with T-rich single-stranded domains. Cleavage reactivity depends strongly on structural context, as strikingly demonstrated with DNA substrates of the form (GGGTTA)n. This repeat sequence from the human telomere is known to switch between parallel and antiparallel G-quadruplex (G4) topologies with a change from potassium to sodium buffer: notably, bPNA-copper complexes efficiently cleave long repeat sequences into ∼22-nucleotide portions in sodium, but not potassium, buffer. We hypothesize preferential cleavage of the antiparallel topology (Na+) over the parallel topology (K+) due to the greater accessibility of the TTA loop to bPNA in the antiparallel (Na+) form. Similar ion-sensitive telomere shortening upon treatment with bPNA nucleases can be observed in both isolated and intracellular DNA from PC3 cells by quantitative polymerase chain reaction. Live cell treatment was accompanied by accelerated cellular senescence, as expected for significant telomere shortening. Taken together, the loop-targeting approach of bPNA chemical nucleases complements prior intercalation strategies targeting duplex and quadruplex DNA. Structurally sensitive loop targeting enables discrimination between similar target sequences, thus expanding bPNA targeting beyond simple oligo-T sequences. In addition, bPNA nucleases are cell membrane permeable and therefore may be used to target native intracellular substrates. In addition, these data indicate that bPNA scaffolds can be a platform for new synthetic binders to particular nucleic acid structural motifs.

Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains.

We report herein the synthesis and DNA/RNA binding properties of bPNA+, a new variant of bifacial peptide nucleic acid (bPNA) that binds oligo T/U nucleic acids to form triplex hybrids. By virtue of a new bivalent side chain on bPNA+, similar DNA affinity and hybrid thermostability can be obtained with half the molecular footprint of previously reported bPNA. Lysine derivatives bearing two melamine bases (K2M) can be prepared on multigram scale by double reductive alkylation with melamine acetaldehyde, resulting in a tertiary amine side chain that affords both peptide solubility and selective base-triple formation with 4 T/U bases; the Fmoc-K2M derivative can be used directly in solid phase peptide synthesis, rendering bPNA+ conveniently accessible. A compact bPNA+binding site of two U6 domains can be genetically encoded to replace existing 6 bp stem elements at virtually any location within an RNA transcript. We thus replaced internal 6 bp RNA stems that supported loop regions with 6 base-triple hybrid stems using fluorophore-labeled bPNA+. As the loop regions engaged in RNA tertiary interactions, the labeled hybrid stems provided a fluorescent readout; bPNA+ enabled this readout without covalent chemical modification or introduction of new structural elements. This strategy was demonstrated to be effective for reporting on widely observed RNA tertiary interactions such as intermolecular RNA-RNA kissing loop dimerization, RNA-protein binding, and intramolecular RNA tetraloop-tetraloop receptor binding, illustrating the potential general utility of this method. The modest 6 bp stem binding footprint of bPNA+ makes the hybrid stem replacement method practical for noncovalent installation of synthetic probes of RNA interactions. We anticipate that bPNA+ structural probes will be useful for the study of tertiary interactions in long noncoding RNAs.

Triplex Hybridization of siRNA with Bifacial Glycopolymer Nucleic Acid Enables Hepatocyte-Targeted Silencing.

Herein, we describe a versatile non-covalent strategy for packaging nucleic acid cargo with targeting modalities, based on triplex hybridization of oligo-uridylate RNA with bifacial polymer nucleic acid (bPoNA). Polyacrylate bPoNA was prepared and side chain-functionalized with N-acetylgalactosamine (GalNAc), which is known to enable delivery to hepatocytes and liver via binding to the asialoglycoprotein receptor (ASGPR). Polymer binding resulted in successful delivery of both native and synthetically modified siRNAs to HepG2 cells in culture, yielding in low nanomolar IC50 silencing of the endogenous ApoB target, in line with observations of expected Dicer processing of the polymer-siRNA targeting complex. Indeed, in vitro Dicer treatment of the polymer complex indicated that triplex hybridization does not impede RNA processing and release from the polymer. The complex itself elicited a quiescent immunostimulation profile relative to free RNA in a cytokine screen, setting the stage for a preliminary in vivo study in a high-calorie-diet mouse model. Gratifyingly, we observed significant ApoB silencing in a preliminary animal study, validating bPoNA as an in vivo carrier platform for systemic siRNA delivery. Thus, this new siRNA carrier platform exhibits generally useful function and is accessible through scalable synthesis. In addition to its utility as a carrier, the triplex-hybridizing synthetic platform could be useful for optimization screens of siRNA sequences using the identical polymer carriers, thus alleviating the need for covalent ligand modification of each RNA substrate.

Small Molecule Recognition Triggers Secondary and Tertiary Interactions in DNA Folding and Hammerhead Ribozyme Catalysis.

We have identified tris(2-aminoethyl)amine (tren)-derived scaffolds with two (t2M) or four (t4M) melamine rings that can target oligo T/U domains in DNA/RNA. Unstructured T-rich DNAs cooperatively fold with the tren derivatives to form hairpin-like structures. Both t2M and t4M act as functional switches in a family of hammerhead ribozymes deactivated by stem or loop replacement with a U-rich sequence. Catalysis of bond scission in these hammerhead ribozymes could be restored by putative t2M/t4M refolding of stem secondary structure or tertiary bridging interactions between loop and stem. The simplicity of the t2M/t4M binding site enables programming of allostery in RNAs, recoding oligo-U domains as potential sites for secondary structure or tertiary contact. In combination with a facile and general method for installation of the t2M motif on primary amines, the method described herein streamlines design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes.