RESUMEN
We report herein a study on the impact of bifacial peptide nucleic acid (bPNA) amino acid composition and backbone modification on DNA binding. A series of bPNA backbone variants with identical net charge were synthesized to display either 4 or 6 melamine (M) bases. These bases form thymine-melamine-thymine (TMT) base-triples, resulting in triplex hybrid stem structures with T-rich DNAs. Analyses of 6â M bPNA-DNA hybrids suggested that hybrid stability was linked to amino acid secondary structure propensities, prompting a more detailed study in shorter 4â M bPNAs. We synthesized 4â M bPNAs predisposed to adopt helical secondary structure via helix-turn nucleation in 7-residue bPNAs using double-click covalent stapling. Generally, hybrid stability improved upon stapling, but amino acid composition had a more significant effect. We also pursued an alternative strategy for bPNA structural preorganization by incorporation of residues with strong backbone amide conformational preferences such as 4R- and 4S-fluoroprolines. Notably, these derivatives exhibited an additional improvement in hybrid stability beyond both unsubstituted proline bPNA analogues and the helically patterned bPNAs. Overall, these findings demonstrate the tunability of bPNA-DNA hybrid stability through bPNA backbone structural propensities and amino acid composition.
Asunto(s)
Ácidos Nucleicos de Péptidos , Aminoácidos , ADN/química , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Timina/químicaRESUMEN
General design principles for recognition at noncanonical interfaces of DNA and RNA remain elusive. Triplex hybridization of bifacial peptide nucleic acids (bPNAs) with oligo-T/U DNAs and RNAs is a robust recognition platform that can be used to define structure-function relationships in synthetic triplex formation. To this end, a set of minimal (mw < 1 kD) bPNA variants was synthesized to probe the impact of amino acid secondary structural propensity, stereochemistry, and backbone cyclization on hybridization with short, unstructured T-rich DNA and U-rich RNAs. Thermodynamic parameters extracted from optical melting analyses of bPNA variant hybrids indicated that there are two bPNA backbone modifications that significantly improve hybridization: alternating (d, l) configuration in open-chain dipeptides and homochiral dipeptide cyclization to diketopiperazine. Further, binding to DNA is preferred over RNA for all bPNA variants. Thymine-uracil substitutions in DNA substrates revealed that the methyl group of thymine accounts for 71% of ΔΔGDNA-RNA for open-chain bPNAs but only 40% of ΔΔGDNA-RNA for diketopiperazine bPNA, suggesting a greater sensitivity to RNA conformation and more optimized stacking in the cyclic bPNA. Together, these data reveal pressure points for tuning triplex hybridization at the chiral centers of bPNA, backbone conformation, stacking effects at the base triple, and the nucleic acid substrate itself. A structural blueprint for enhancing bPNA targeting of both DNA and RNA substrates includes syndiotactic base presentation (as found in homochiral diketopiperazines and d, l peptides), expansion of base stacking, and further investigation of bPNA backbone preorganization.
Asunto(s)
ADN/química , Ácidos Nucleicos de Péptidos/química , ARN/química , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Termodinámica , Timina/química , Uracilo/químicaRESUMEN
We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3' oligo-A tail of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs composed of α-linked peptide, isodipeptide, and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE half-life plunging to 6 min from >24 h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinities, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 > 4000× , supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for the investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.
Asunto(s)
Ácidos Nucleicos de Péptidos/farmacología , ARN Largo no Codificante/efectos de los fármacos , Línea Celular Tumoral , Exonucleasas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Conformación de Ácido Nucleico , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismoRESUMEN
The notion of using synthetic heterocycles instead of the native bases to interface with DNA and RNA has been explored for nearly 60 years. Unnatural bases compatible with the DNA/RNA coding interface have the potential to expand the genetic code and co-opt the machinery of biology to access new macromolecular function; accordingly, this body of research is core to synthetic biology. While much of the literature on artificial bases focuses on code expansion, there is a significant and growing effort on docking synthetic heterocycles to noncoding nucleic acid interfaces; this approach seeks to illuminate major processes of nucleic acids, including regulation of transcription, translation, transport, and transcript lifetimes. These major avenues of research at the coding and noncoding interfaces have in common fundamental principles in molecular recognition. Herein, we provide an overview of foundational literature in biophysics of base recognition and unnatural bases in coding to provide context for the developing area of targeting noncoding nucleic acid interfaces with synthetic bases, with a focus on systems developed through iterative design and biophysical study.
Asunto(s)
ADN/metabolismo , ARN/metabolismo , Emparejamiento Base , ADN/química , Enlace de Hidrógeno , Nucleósidos de Purina/química , Nucleósidos de Purina/metabolismo , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/metabolismo , ARN/química , Biología Sintética/métodosRESUMEN
We report herein a dual-purpose role for polyacidic domains in an aqueous-phase polymer amphiphile assembly. In addition to their typical role as ionized water-solubilizing and self-repulsive motifs, we find that polycarboxylic acid domains uniquely enable high levels of hydrophobic drug encapsulation. By attenuated total reflectance infrared spectroscopy, we find significant differences in the carbonyl stretching region of the nanoparticles formed by polyacidic amphiphiles relative to those in soluble, single-domain poly(acrylic acid), suggesting that stabilization may be derived from limited ionization of the carboxylate groups upon assembly. Acidic-hydrophobic diblock polyacrylates were prepared and coassembled with up to 60 wt % camptothecin (CPT) into nanoparticles, the highest loading reported to date. Controlled release of bioactive CPT from polymer nanoparticles is observed, as well as protection from human serum albumin-induced hydrolysis. Surface protection with PEG limits uptake of the CPT-loaded nanoparticles by MCF-7 breast cancer cells, as expected. Acidic-hydrophobic polymer amphiphiles thus have the hallmarks of a useful and general drug delivery platform and are readily accessible from living radical polymerization of cheap, commercially available monomers. We highlight here the potential utility of this common polymer design in high-capacity, controlled-release polymer nanoparticle systems.