Telomere Length Measurement in Human Tissue Sections by Quantitative Fluorescence In Situ Hybridization (Q-FISH).

Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections.

Peptide nucleic acids can form hairpins and bind RNA-binding proteins.

RNA-binding proteins (RBPs) are a major class of proteins that interact with RNAs to change their fate or function. RBPs and the ribonucleoprotein complexes they constitute are involved in many essential cellular processes. In many cases, the molecular details of RBP:RNA interactions differ between viruses, prokaryotes and eukaryotes, making prokaryotic and viral RBPs good potential drug targets. However, targeting RBPs with small molecules has so far been met with limited success as RNA-binding sites tend to be extended, shallow and dynamic with a mixture of charged, polar and hydrophobic interactions. Here, we show that peptide nucleic acids (PNAs) with nucleic acid-like binding properties and a highly stable peptide-like backbone can be used to target some RBPs. We have designed PNAs to mimic the short RNA stem-loop sequence required for the initiation of prokaryotic signal recognition particle (SRP) assembly, a target for antibiotics development. Using a range of biophysical and biochemical assays, the designed PNAs were demonstrated to fold into a hairpin structure, bind the targeted protein and compete with the native RNA hairpin to inhibit SRP formation. To show the applicability of PNAs against other RBPs, a PNA was also shown to bind Nsp9 from SARS-CoV-2, a protein that exhibits non-sequence-specific RNA binding but preferentially binds hairpin structures. Taken together, our results support that PNAs can be a promising class of compounds for targeting RNA-binding activities in RBPs.

A Facile Synthesis of Red-Shifted Bis-Quinoline (BisQ) Surrogate Base.

Forced intercalation peptide nucleic acids (FIT-PNAs) are DNA mimics that act as RNA sensors. The sensing event occurs due to sequence-specific RNA hybridization, leading to a substantial increase in fluorescence. The fluorophore in the FIT-PNA is termed a surrogate base. This molecule typically replaces a purine in the PNA sequence. BisQ is a surrogate base that connects two quinolines via a monomethine bond. BisQ-based FIT-PNAs have excellent biophysical features that include high brightness and red-shifted emission (λem, max = 613 nm). In this report, we detail two chemical approaches that allow for the facile synthesis of the BisQ PNA monomer. In both cases, the key compound used for the synthesis of BisQ-CH2COOH is the tBu-ester-modified quinoline synthon (compound 5). Subsequently, one method uses the Alloc acid-protected PNA backbone, whereas the other uses the tBu ester-protected PNA backbone. In the latter case, the overall yield for BisQ acid (compound 7) and BisQ PNA monomer syntheses was 61% in six synthetic steps. This is a substantial improvement to the published procedures to date (7% total yield). Lastly, we have prepared an 11-mer FIT-PNA with either BisQ or thiazole orange (TO) and studied their photophysical properties. We find superior photophysical properties for the BisQ FIT-PNA in terms of the brightness and selectivity, highlighting the added value of using this surrogate base for RNA sensing.

Fusion of FokI and catalytically inactive prokaryotic Argonautes enables site-specific programmable DNA cleavage.

Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, and robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif, but their lack of intrinsic dsDNA unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage, and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo.

Peptide nucleic acid-clicked Ti3C2Tx MXene for ultrasensitive enzyme-free electrochemical detection of microRNA biomarkers.

We report the engineering and synthesis of peptide nucleic acid-functionalized Ti3C2Tx MXene nanosheets as a novel transducing material for amplification-free, nanoparticle-free, and isothermal electrochemical detection of microRNA biomarkers. Through bio-orthogonal copper-free click chemistry, azido-modified MXene nanosheets are covalently functionalized with clickable peptide nucleic acid probes targeting prostate cancer biomarker hsa-miR-141. The platform demonstrates a wide dynamic range, single-nucleotide specificity, and 40 aM detection limit outperforming more complex, amplification-based methods. Its versatility, analytical performance, and stability under serum exposure highlight the immense potential of this first example of click-conjugated MXene in the next generation of amplification-free biosensors.

Repurposing an antimicrobial peptide for the development of a dual ion channel/molecular receptor-like platform for metal ion detection.

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.

Effective lowering of α-synuclein expression by targeting G-quadruplex structures within the SNCA gene.

Alpha-synuclein, encoded by the SNCA gene, is a pivotal protein implicated in the pathogenesis of synucleinopathies, including Parkinson's disease. Current approaches for modulating alpha-synuclein levels involve antisense nucleotides, siRNAs, and small molecules targeting SNCA's 5'-UTR mRNA. Here, we propose a groundbreaking strategy targeting G-quadruplex structures to effectively modulate SNCA gene expression and lowering alpha-synuclein amount. Novel G-quadruplex sequences, identified on the SNCA gene's transcription starting site and 5'-UTR of SNCA mRNAs, were experimentally confirmed for their stability through biophysical assays and in vitro experiments on human genomic DNA. Biological validation in differentiated SH-SY5Y cells revealed that well-known G-quadruplex ligands remarkably stabilized these structures, inducing the modulation of SNCA mRNAs expression, and the effective decrease in alpha-synuclein amount. Besides, a novel peptide nucleic acid conjugate, designed to selectively disrupt of G-quadruplex within the SNCA gene promoter, caused a promising lowering of both SNCA mRNA and alpha-synuclein protein. Altogether our findings highlight G-quadruplexes' key role as intriguing biological targets in achieving a notable and successful reduction in alpha-synuclein expression, pointing to a novel approach against synucleinopathies.

Pitfalls and challenges of peptide nucleic acid immobilisation on carbon surfaces for sequence-specific capturing of nucleic acid biomarkers.

Nucleic acid sensors based on a peptide nucleic acid (PNA) probe have seen a surge in interest since their discovery in the 1990s, and after the patent protecting them expired in 2013. The appeal of PNA as capture and/or sensing probes as an alternative to standard DNA or RNA oligonucleotides originates from their superior chemical stability and affinity for complementary oligonucleotides, as well as their increased responsiveness to single base mismatches. The implementation of PNA probes onto optical and electrochemical sensors has showed great promise although progress has been hampered by issues mostly associated with surface chemistry, probe accessibility and non-specific binding. Herein, we report on a systematic comparison between various PNA immobilisation strategies on carbon substrates based on both covalent and non-covalent chemistries. Besides the use of standard electrochemical techniques to characterise the extent of surface modification, the ability of immobilised PNAs to engage in chemical interactions with freely diffusing molecules was also investigated. Using original chemical tags, this study provides a unique insight into the impact of immobilisation chemistries on PNA's (bio)availability. Rapid immobilisation of biotinylated PNA oligomers on screen-printed carbon electrode (SPCE) coated with adsorbed polystreptavidin (pSA) demonstrated highest efficiency and ease in the preparation process. An original nucleic acid sensor using this immobilisation chemistry is reported that is based on a sandwich assay between a surface bound PNA capture probe and a freely diffusing electrochemically active PNA sensing probe.

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses.

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43-25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

Supramolecular polyplexes from Janus peptide nucleic acids (bm-PNA-G5): self-assembled bm-PNA G-quadruplex and its tetraduplex with DNA.

Nucleic acids (DNA and RNA) can form diverse secondary structures ranging from hairpins to duplex, triplex, G4-tetraplex and C4-i-motifs. Many of the DNA analogues designed as antisense oligonucleotides (ASO) are also adept at embracing such folded structures, although to different extents with altered stabilities. One such analogue, peptide nucleic acid (PNA), which is uncharged and achiral, forms hybrids with complementary DNA/RNA with greater stability and specificity than DNA:DNA/RNA hybrids. Like DNAs, these single-stranded PNAs can form PNA:DNA/RNA duplexes, PNA:DNA:PNA triplexes, PNA-G4 tetraplexes and PNA-C4-i-motifs. We have recently designed Janus-like bimodal PNAs endowed with two different nucleobase sequences on either side of a single aminoethylglycyl (aeg) PNA backbone and shown that these can simultaneously bind to two complementary DNA sequences from both faces of PNA. This leads to the formation of supramolecular polyplexes such as double duplexes, triple duplexes and triplexes of double duplexes with appropriate complementary DNA/RNA. Herein, we demonstrate that Janus/bimodal PNA with a poly G-sequence on the triazole side of the PNA backbone and mixed bases on the t-amide side, templates the initial formation of a (PNA-G5)4 tetraplex (triazole side), followed by the formation of a PNA:DNA duplex (t-amide side). Such a polyplex shows synergistic overall stabilisation compared to the isolated duplexes/quadruplex. The assembly of polyplexes with a shared backbone for duplexes and tetraplexes is programmable and may have potential applications in the self-assembly of nucleic acid nano- and origami structures. It is also shown that Janus PNAs enter the cells better than the standard aeg-PNA oligomers, and hence have implications for in vivo applications as well.

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure.

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

Engineering of a DNA/γPNA Hybrid Nanoreporter for ctDNA Mutation Detection via γPNA Urinalysis.

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid) hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations.

Electrophoretically Snagging Viral Genomes in Wormlike Micelle Networks Using Peptide Nucleic Acid Amphiphiles and dsDNA Oligomers.

We demonstrate that the attachment of 30-170 bp dsDNA oligomers to ssDNA viral genomes gives a significant additional mobility shift in micelle-tagging electrophoresis (MTE). In MTE, a modified peptide nucleic acid amphiphile is attached to the viral genome to bind drag-inducing micelles present in capillary electrophoresis running buffers. Further attachment of 30-170 bp dsDNA oligomers drastically shifts the mobility of the 5.1 kB ssDNA genome of mouse minute virus (MMV), providing a new mechanism to improve resolution in CE-based analysis of kilobase nucleic acids. A model based on biased-reptation electrophoresis, end-labeled free-solution electrophoresis, and Ferguson gel-filtration theory is presented to describe the observed mobility shifts.

Papain-Mediated Conjugation of Peptide Nucleic Acids to Delivery Peptides: A Density Functional Theory/Molecular Mechanics Metadynamics Study in Aqueous and Organic Solvent.

Enzymatic peptide synthesis is a powerful alternative to solid-phase methods, as enzymes can have high regio- and stereoselectivity and high yield and require mild reaction conditions. This is beneficial in formulation research due to the rise of nucleic acid therapies. Peptide nucleic acids (PNAs) have a high affinity toward DNA and RNA, and their solubility and cellular delivery can be improved via conjugation to peptides. Here, we designed and assessed the viability of the papain enzyme to conjugate four PNA-peptide models in water and an organic solvent using QM/MM metadynamics. We found that the reactions in water yield better results, where three conjugates could potentially be synthesized by the enzyme, with the first transition state as the rate-limiting step, with an associated energy of 14.53 kcal mol-1, although with a slight endergonic profile. The results highlight the importance of considering the enzyme pockets and different substrate acceptivities and contribute to developing greener, direct, and precise synthetic routes for nucleic acid-based therapies. By exploring the enzyme's potential in conjunction with chemical synthesis, current protocols can be simplified for the synthesis of longer nucleic acids and peptide sequences (and, by extension, proteins) from smaller oligo or peptide blocks.

Preclinical Pharmacokinetics in Tumors and Normal Tissues of the Antigene PNA Oligonucleotide MYCN-Inhibitor BGA002.

Although MYCN has been considered an undruggable target, MYCN alterations confer poor prognosis in many pediatric and adult cancers. The novel MYCN-specific inhibitor BGA002 is an antigene peptide nucleic acid oligonucleotide covalently bound to a nuclear localization signal peptide. In the present study, we characterized the pharmacokinetics (PK) of BGA002 after single and repeated administration to mice using a novel specific enzyme-linked immunosorbent assay. BGA002 concentrations in plasma showed linear PK, with dose proportional increase across the tested dose levels and similar exposure between male and female and between intravenous and subcutaneous route of administration. Repeated dosing resulted in no accumulation in plasma. Biodistribution up to 7 days after single subcutaneous administration of [14C]-radiolabeled BGA002 showed broad tissues and organ distribution (suggesting a potential capability to reach primary tumor and metastasis in several body sites), with high concentrations in kidney, liver, spleen, lymph nodes, adrenals, and bone marrow. Remarkably, we demonstrated that BGA002 concentrates in tumors after repeated systemic administrations in three mouse models with MYCN amplification (neuroblastoma, rhabdomyosarcoma, and small-cell lung cancer), leading to a significant reduction in tumor weight. Taking into account the available safety profile of BGA002, these data support further evaluation of BGA002 in patients with MYCN-positive tumors.

Use of peptide nucleic acid probe to determine telomere dynamics in improving chromosome analysis in genetic toxicology studies.

Genetic toxicology, strategically located at the intersection of genetics and toxicology, aims to demystify the complex interplay between exogenous agents and our genetic blueprint. Telomeres, the protective termini of chromosomes, play instrumental roles in cellular longevity and genetic stability. Traditionally karyotyping and fluorescence in situ hybridisation (FISH), have been indispensable tools for chromosomal analysis following exposure to genotoxic agents. However, their scope in discerning nuanced molecular dynamics is limited. Peptide Nucleic Acids (PNAs) are synthetic entities that embody characteristics of both proteins and nucleic acids and have emerged as potential game-changers. This perspective report comprehensively examines the vast potential of PNAs in genetic toxicology, with a specific emphasis on telomere research. PNAs' superior resolution and precision make them a favourable choice for genetic toxicological assessments. The integration of PNAs in contemporary analytical workflows heralds a promising evolution in genetic toxicology, potentially revolutionizing diagnostics, prognostics, and therapeutic avenues. In this timely review, we attempted to assess the limitations of current PNA-FISH methodology and recommend refinements.

Impact of charges on the hybridization kinetics and thermal stability of PNA duplexes.

Peptide nucleic acid (PNA) is a prominent artificial nucleic acid mimetic and modifications at the γ-position of the peptidic backbone are known to further enhance the desirable properties of PNA in terms of duplex stability. Here, we leveraged a propargyl ether modification at this position for late stage functionalization of PNA to obtain positively charged (cationic amino and guanidinium groups), negatively charged (anionic carboxylate and alkyl phosphonate groups) and neutral (PEG) PNAs to assess the impact of these charges on DNA : PNA and PNA : PNA duplex formation. Thermal stability analysis findings concurred with prior studies showing PNA : DNA duplexes are moderately more stable with cationic PNAs than anionic PNAs at physiological salt concentrations. We show that this effect is derived predominantly from differences in the association kinetics. For PNA : PNA duplexes, anionic PNAs were found to form the most stable duplexes, more stable than neutral PNA : PNA duplexes.

Dynamics of terminal fraying-peeling and hydrogen bonds dictates the sequential vs. cooperative melting pathways of nanoscale DNA and PNA triplexes.

Peptide nucleic acids (PNAs) are charge-neutral synthetic DNA/RNA analogues. In many aspects of biology and biotechnology, the details of DNA and PNA melting reaction coordinates are crucial, and their associative/dissociative details remain inadequately understood. In the current study, we have attempted to gain insights into comparative melting pathways and binding affinity of iso-sequences of an 18-mer PNA-DNA-PNA triplex and the analogous DNA-DNA-DNA triplex, and DNA-DNA and PNA-DNA duplexes. It is intriguing that while the DNA-DNA-DNA triplex melts in two sequential steps, the PNA-DNA-PNA triplex melts in a single step and the mechanistic aspects for this difference are still not clear. We report an all-atom molecular dynamics simulation of both complexes in the temperature range of 300 to 500 K with 20 K intervals. Based on the trajectory analysis, we provide evidence that the association and dissociation are dictated by the differences in fraying-peeling effects from either terminus to the center in a zipper pattern among the PNA-DNA-PNA triplex and DNA-DNA-DNA triplexes. These are shown to be governed by the different characteristics of H-bonding, RMSD, and Free Energy Landscape (FEL) as analyzed by PCA, leading to the DNA-DNA-DNA triplex exhibiting sequential melting, while the PNA-DNA-PNA triplex shows cooperative melting of the whole fragment in a single-step. The PNA-DNA-PNA triplex base pairs are thermodynamically more stable than the DNA-DNA-DNA triplex, with the binding affinity of PNA-TFO to the PNA : DNA duplex being higher than that of DNA-TFO to the DNA : DNA duplex. The investigation of the association/dissociation of PNA-TFO to the PNA-DNA duplex has relevance and importance in the emerging effective applications of oligonucleotide therapy.

Hybrid PNA-peptide hydrogels as injectable CEST-MRI agents.

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH2, named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs).

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.