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.
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.
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.
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.
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