Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA

Brodyagin, Nikita; Hnedzko, Dziyana; Mackay, James; Rozners, Eriks

doi:10.1007/978-1-0716-0243-0_9

Cited by 15 publications

(29 citation statements)

References 56 publications

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“…We started our study by screening all possible variants of six-membered heterocyclic nucleobases containing one (P 1 –P 3 , pyridine) and two (P 4 –P 9 , pyrimidine, pyrazine, and pyridazine) nitrogen atoms using the model system of 9-mer PNA N and RNA hairpin HRP C (Figure ), as in our previous studies. − , …”

Section: Resultsmentioning

confidence: 99%

“…Binding affinity of P N -modified PNAs was measured using isothermal titration calorimetry (ITC, as in our previous studies − , ) and UV thermal melting at 300 nm. The latter reported specifically on the triple helix formation by measuring changes in absorbance at a wavelength (300 nm) unique to the M nucleobases (for structure of M, see Figure ).…”

Section: Resultsmentioning

confidence: 99%

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Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA

et al. 2021

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“…The M monomer and J monomer , were synthesized using previously reported literature procedures. Solid-phase synthesis of PNA was performed on an Expedite 8909 synthesizer at a 2 μmol scale using NovaSyn TG Sieber resin (Novabiochem) as previously reported by our group . PNAs were cleaved from the solid support using 0.6 mL of 20% m -cresol in TFA for 2.0 h using a two-syringe pull–push method .…”

Section: Methodsmentioning

confidence: 99%

“…Solid-phase synthesis of PNA was performed on an Expedite 8909 synthesizer at a 2 μmol scale using NovaSyn TG Sieber resin (Novabiochem) as previously reported by our group. 30 PNAs were cleaved from the solid support using 0.6 mL of 20% m-cresol in TFA for 2.0 h using a two-syringe pull− push method. 30 The cleavage cocktail was distributed into three Eppendorf tubes and precipitated by the addition of chilled diethyl ether (∼1.0 mL) followed by the centrifugation (15000 rpm).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…30 PNAs were cleaved from the solid support using 0.6 mL of 20% m-cresol in TFA for 2.0 h using a two-syringe pull− push method. 30 The cleavage cocktail was distributed into three Eppendorf tubes and precipitated by the addition of chilled diethyl ether (∼1.0 mL) followed by the centrifugation (15000 rpm). The crude PNA was dissolved in purified water (∼1.0 mL) and analyzed using a Shimadzu LC-MS 2020 single quadrupole instrument.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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The 2-Aminopyridine Nucleobase Improves Triple-Helical Recognition of RNA and DNA When Used Instead of Pseudoisocytosine in Peptide Nucleic Acids

et al. 2021

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Pseudoisocytosine (J), a neutral analogue of protonated cytosine, is currently the gold standard modified nucleobase in peptide nucleic acids (PNAs) for the formation of J•G-C triplets that are stable at physiological pH. This study shows that triple-helical recognition of RNA and DNA is significantly improved by using 2-aminopyridine (M) instead of J. The positively charged M forms 3-fold stronger M+•G-C triplets than J with uncompromised sequence selectivity. Replacement of six Js with Ms in a PNA 9-mer increased its binding affinity by ∼2 orders of magnitude. Mmodified PNAs prefer binding double-stranded RNA over DNA and disfavor off-target binding to single-stranded nucleic acids. Taken together, the results show that M is a promising modified nucleobase that significantly improves triplex-forming PNAs and may provide breakthrough developments for therapeutic and biotechnology applications.

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Improved Triplex‐Forming Isoorotamide PNA Nucleobases for A−U Recognition of RNA Duplexes**

Talbott,

Tessier,

Harding

et al. 2023

Chemistry A European J

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Four new isoorotamide (Io)‐containing PNA nucleobases have been designed for A−U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A−U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A‐binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A‐binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.

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Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA

Cited by 15 publications

References 56 publications

Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA

Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA

The 2-Aminopyridine Nucleobase Improves Triple-Helical Recognition of RNA and DNA When Used Instead of Pseudoisocytosine in Peptide Nucleic Acids

Improved Triplex‐Forming Isoorotamide PNA Nucleobases for A−U Recognition of RNA Duplexes**

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