Red-emitting FIT-PNAs: “On site” detection of RNA biomarkers in fresh human cancer tissues

Hashoul, Dina; Shapira, Rachel; Falchenko, Maria; Tepper, Odelia; Paviov, Vera; Nissan, Aviram; Yavin, Eylon

doi:10.1016/j.bios.2019.04.056

Cited by 13 publications

(9 citation statements)

References 45 publications

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“…As an additional control, dK 4 –PNA was also prepared with the scrambled PNA sequence (Table ). dK 4 –PNA conjugates have also been previously reported as cancer cell-permeable at a 1 μM concentration . Fluorescein isothiocyanate (FITC) labeling at the N-terminus of both C 9 –PNA and dK 4 –PNA was carried out to allow us to follow the cellular uptake of both PNAs that differ only in their peptide sequence (Table ).…”

Section: Resultsmentioning

confidence: 99%

AntimiR-155 Cyclic Peptide–PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity

et al. 2019

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Efficient delivery of nucleic acids into cells still remains a great challenge. Peptide nucleic acids (PNAs) are DNA analogues with a neutral backbone and are synthesized by solid phase peptide chemistry. This allows a straightforward synthetic route to introduce a linear short peptide (a.k.a. cell-penetrating peptide) to the PNA molecule as a means of facilitating cellular uptake of PNAs. Herein, we have devised a synthetic route in which a cyclic peptide is prepared on a solid support and is extended with the PNA molecule, where all syntheses are accomplished on the solid phase. This allows the conjugation of the cyclic peptide to the PNA molecule with the need of only one purification step after the cyclic peptide–PNA conjugate (C 9 –PNA) is cleaved from the solid support. The PNA sequence chosen is an antimiR-155 molecule that is complementary to mature miR-155, a well-established oncogenic miRNA. By labeling C 9 –PNA with fluorescein isothiocyanate, we observe efficient cellular uptake into glioblastoma cells (U87MG) at a low concentration (0.5 μM), as corroborated by fluorescence-activated cell sorting (FACS) analysis and confocal microscopy. FACS analysis also suggests an uptake mechanism that is energy-dependent. Finally, the antimiR activity of C 9 –PNA was shown by analyzing miR155 levels by quantitative reverse transcription polymerase chain reaction and by observing a reduction in cell viability and proliferation in U87MG cells, as corroborated by XTT and colony formation assays. Given the added biological stability of cyclic versus linear peptides, this synthetic approach may be a useful and straightforward approach to synthesize cyclic peptide–PNA conjugates.

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Section: Resultsmentioning

confidence: 99%

AntimiR-155 Cyclic Peptide–PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity

et al. 2019

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“…Six K13 FIT-PNA probes (Table 1) were previously synthesized and fully characterized by HPLC and ESI-MS. 51 WT K13 FIT-PNAs (1 and 2), K67T FIT-PNA, and NT FIT-PNA were synthesized on the solid support (Novasyn TGA resin, 0.25 mmol/g) by standard peptide chemistry using Fmoc-protected PNA monomers. 52 HPLC and ESI-MS of these FIT-PNAs are shown in the Supporting Information (Figures S1−S8).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

FIT-PNAs as RNA-Sensing Probes for Drug-Resistant Plasmodium falciparum

Tepper

Peled

Fastman³

et al. 2022

ACS Sens.

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Detecting RNA at single-nucleotide resolution is a formidable task. Plasmodium falciparum is the deadliest form of malaria in humans and has shown to gain resistance to essentially all antimalarial drugs including artemisinin and chloroquine. Some of these drug resistances are associated with single-nucleotide polymorphisms (SNPs). Forced-intercalation peptide nucleic acids (FIT-PNAs) are DNA mimics that are designed as RNA-sensing molecules that fluoresce upon hybridization to their complementary (RNA) targets. We have previously designed and synthesized FIT-PNAs that target the C580Y SNP in the K13 gene of P. falciparum. In addition, we have now prepared FIT-PNAs that target the K76T SNP in the CRT gene of P. falciparum. Both SNPs are common ones associated with artemisinin and chloroquine drug resistance, respectively. Our FIT-PNAs are conjugated to a simple cell-penetrating peptide (CPP) that consists of eight D-lysines (dK 8 ), which renders these FIT-PNAs cell-permeable to infected red blood cells (iRBCs). Herein, we demonstrate that FIT-PNAs clearly discriminate between wild-type (WT) strains (NF54-WT: artemisinin-sensitive or chloroquine-sensitive) and mutant strains (NF54-C580Y: artemisinin-resistant or Dd2: chloroquine-resistant) of P. falciparum parasites. Simple incubation of FIT-PNAs with live blood-stage parasites results in a substantial difference in fluorescence as corroborated by FACS analysis and confocal microscopy. We foresee FIT-PNAs as molecular probes that will provide a fast, simple, and cheap means for the assessment of drug resistance in malariaa tool that would be highly desirable for the optimal choice of antimalarial treatment in endemic countries.

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“…BisQ-modified PNAs have been used to detect KRT20 mRNA, which is over expressed in colorectal cancer, in live cancer cells [260]. An 18-mer BisQ-modified PNA displayed 20-fold fluorescence enhancement in the presence of the target sequence while a scrambled PNA sequence containing BisQ gave no fluorescence response.…”

Section: Detection Of Dna and Mrnamentioning

confidence: 99%

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

Brodyagin

Katkevičs

Kotikam

et al. 2021

Beilstein J. Org. Chem.

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Peptide nucleic acid (PNA) is arguably one of the most successful DNA mimics, despite a most dramatic departure from the native structure of DNA. The present review summarizes 30 years of research on PNA’s chemistry, optimization of structure and function, applications as probes and diagnostics, and attempts to develop new PNA therapeutics. The discussion starts with a brief review of PNA’s binding modes and structural features, followed by the most impactful chemical modifications, PNA enabled assays and diagnostics, and discussion of the current state of development of PNA therapeutics. While many modifications have improved on PNA’s binding affinity and specificity, solubility and other biophysical properties, the original PNA is still most frequently used in diagnostic and other in vitro applications. Development of therapeutics and other in vivo applications of PNA has notably lagged behind and is still limited by insufficient bioavailability and difficulties with tissue specific delivery. Relatively high doses are required to overcome poor cellular uptake and endosomal entrapment, which increases the risk of toxicity. These limitations remain unsolved problems waiting for innovative chemistry and biology to unlock the full potential of PNA in biomedical applications.

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Red-emitting FIT-PNAs: “On site” detection of RNA biomarkers in fresh human cancer tissues

Cited by 13 publications

References 45 publications

AntimiR-155 Cyclic Peptide–PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity

AntimiR-155 Cyclic Peptide–PNA Conjugate: Synthesis, Cellular Uptake, and Biological Activity

FIT-PNAs as RNA-Sensing Probes for Drug-Resistant Plasmodium falciparum

Chemical approaches to discover the full potential of peptide nucleic acids in biomedical applications

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