RESUMEN
Peptide nucleic acid (PNA) is emerging as a promising ligand for triple-helical recognition of folded biologically relevant RNA. Chemical modifications are actively being developed to achieve high affinity and sequence specificity under physiological conditions. In this study, we compared two modified PNA nucleobases, 2-aminopyridine (M) and 4-thiopseudisocytosine (L), as alternatives to protonated cytosine (unfavorable under physiological conditions), to form more stable triplets than C+·G-C. Both nucleobases formed M+·G-C and L·G-C triplets of similar stability; however, the L-modified PNAs showed somewhat reduced sequence specificity. In conclusion, M and L represent two alternative solutions to the problem of cytosine protonation in triple-helical recognition of RNA. In M, the pKa is increased to favor partial protonation, which improves solubility and cellular uptake of M-modified PNAs. In L, the sulfur substitution enhances favorable hydrophobic interactions, which may have advantages in avoiding off-target effects that may be caused by cationic modifications. However, our results showed that substituting Ms with Ls did not restore the sequence specificity of a PNA containing cationic groups.
RESUMEN
Triple-helical recognition of any sequence of double-stranded RNA requires high affinity Hoogsteen hydrogen binding to pyrimidine interruptions of polypurine tracts. Because pyrimidines have only one hydrogen bond donor/acceptor on Hoogsteen face, their triple-helical recognition is a formidable problem. The present study explored various five-membered heterocycles and linkers that connect nucleobases to backbone of peptide nucleic acid (PNA) to optimize formation of Xâ¢C-G and Yâ¢U-A triplets. Molecular modeling and biophysical (UV melting and isothermal titration calorimetry) results revealed a complex interplay between the heterocyclic nucleobase and linker to PNA backbone. While the five-membered heterocycles did not improve pyrimidine recognition, increasing the linker length by four atoms provided promising gains in binding affinity and selectivity. The results suggest that further optimization of heterocyclic bases with extended linkers to PNA backbone may be a promising approach to triple-helical recognition of RNA.
Asunto(s)
Ácidos Nucleicos de Péptidos , Ácidos Nucleicos de Péptidos/química , Conformación de Ácido Nucleico , ARN Bicatenario , Pirimidinas , Modelos MolecularesRESUMEN
Cell-penetrating peptides (CPPs) have been extensively used to deliver peptide nucleic acid (PNA) in cells. We have previously found that replacement of cytosine in triplex-forming PNAs with 2-aminopyridine (M) not only enhanced RNA binding, but also improved cellular uptake of PNAs. In this study, we used confocal fluorescence microscopy to evaluate the ability of CPPs to further improve cellular uptake of M-modified PNAs. We found that PNAs conjugated with Tat and octa-arginine peptides were effectively taken up in MCF7 cells when supplied in cell media at 1 µM. Remarkably, M-modified PNA without any CPP conjugation also showed strong uptake when the concentration was increased to 5 µM. Majority of PNA conjugates remained localized in distinct cytoplasmic vesicles, as judged by dot-like fluorescence patterns. However, M-modified PNAs conjugated with Tat, octa-arginine, and even a simple tri-lysine peptide also showed dispersed fluorescence in cytoplasm and were taken up in nuclei where they localized in larger vesicles, most likely nucleoli. Endosomolytic peptides or chemicals (chloroquine and CaCl2 ) did not release the conjugates from cytosolic vesicles, which suggested that the PNAs were not entrapped in endosomes. We hypothesize that M-modified PNAs escape endosomes and accumulate in cellular compartments rich in RNA, such as nucleoli, stress granules, and P-bodies.
Asunto(s)
Péptidos de Penetración Celular , Ácidos Nucleicos de Péptidos , Aminopiridinas , Arginina , Células HeLa , Humanos , Ácidos Nucleicos de Péptidos/metabolismo , ARNRESUMEN
Sequence specific recognition of regulatory noncoding RNAs would open new possibilities for fundamental science and medicine. However, molecular recognition of such complex double-stranded RNA (dsRNA) structures remains a formidable problem. Recently, we discovered that peptide nucleic acids (PNAs) form an unusually stable and sequence-specific triple helix with dsRNA. Triplex-forming PNAs could become universal tools for recognition of noncoding dsRNAs but are limited by the requirement of polypurine tracts in target RNAs as only purines form stable Hoogsteen hydrogen bonded base triplets. Herein, we systematically surveyed simple nitrogen heterocycles PN as modified nucleobases for recognition of cytosine in PN*C-G triplets. We found that a 3-pyridazinyl nucleobase formed significantly more stable PN*C-G triplets than other heterocycles including the pyrimidin-2-one previously used by us and others for recognition of cytosine interruptions in polypurine tracts of PNA-dsRNA triplexes. Our results improve triple helical recognition of dsRNA and provide insights for future development of new nucleobases to expand the sequence scope of noncoding dsRNAs that can be targeted by triplex-forming PNAs.
Asunto(s)
Citosina/química , Ácidos Nucleicos de Péptidos/síntesis química , Purinas/química , Piridazinas/química , ARN Bicatenario/química , Secuencia de Bases , Enlace de Hidrógeno , Conformación Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
Peptide nucleic acids (PNA) with extended isoorotamide containing nucleobases (Io ) were designed for binding A-U base pairs in double-stranded RNA. Isothermal titration calorimetry and UV thermal melting experiments revealed improved affinity for A-U using the Io scaffold in PNA. PNAs having four sequential Io extended nucleobases maintained high binding affinity.
Asunto(s)
Ácidos Nucleicos de Péptidos , Emparejamiento Base , Calorimetría , Conformación de Ácido Nucleico , ARN BicatenarioRESUMEN
Triple-helix formation, using Hoogsteen hydrogen bonding of triplex-forming oligonucleotides, represents an attractive method for sequence-specific recognition of double-stranded nucleic acids. However, practical applications using triple-helix-forming oligonucleotides and their analogues are limited to long homopurine sequences. The key problem for recognition of pyrimidines is that they present only one hydrogen-bond acceptor or donor group in the major groove. Herein, we report our first attempt to overcome this problem by using peptide nucleic acids (PNAs) modified with extended nucleobases that form three hydrogen bonds along the entire Hoogsteen edge of the Watson-Crick base pair. New nucleobase triples (five) were designed, and their hydrogen bonding feasibility was confirmed by ab initio calculations. PNA monomers carrying the modified nucleobases were synthesized and incorporated in short model PNA sequences. Isothermal titration calorimetry showed that these nucleobases had a modest binding affinity for their double-stranded RNA (dsRNA) targets. Finally, molecular modeling of the modified triples in PNA-dsRNA helix suggested that the modest binding affinity was caused by subtle structural deviations from ideal hydrogen-bonding arrangements or disrupted π-stacking of the extended nucleobase scaffolds.