Manual solid-phase synthesis was performed by using 5 mL polyethylene syringe reactors (Phenomenex, Torrance, CA, USA) that are equipped with a fritted disk. RNA oligos were purchased from IDT, USA. Fmoc-PNA monomers were purchased from PolyOrg, Inc. (USA) and used as received. Fmoc-D-Lysine and reagents for solid phase synthesis were purchased from Merck (Germany) and Biolab (Israel). Fmoc-protected cyclopentane PNA monomers (cpG) [60], positively charged guanine (G⁺) [51], and BisQ [61] were synthesized as previously reported.

FIT-PNAs were synthesized on solid phase in a continuous process, thereby eliminating the need for repurification [61]. Coupling of the first monomer, Fmoc-D-Lysine(tBOC)-OH, onto Novasyn TGA Resin was performed as follows: The resin (100 mg, 0.25 mmol/g) was allowed to swell in 2 mL DMF for 2 h. For pre-activation, 5 equivalents of diisopropylcarbodiimide (DIC, 0.125 mmols, 15.8 mg, 19.5 µL), and 0.1 equivalent of 4-dimethylaminopyrimidine (DMAP, 0.0025 mmols, 0.3 mg) were added to a solution of 10 equivalents of Fmoc-D-Lysine(tBOC)-OH (0.25 mmols, 117 mg) in DCM (2.5 mL) in an ice bath. After 20 min, the mixture was evaporated, re-dissolved in dry DMF and added to the resin. After 5h, the resin was washed with dichloromethane (5 × 2 mL), DMF (5 × 2 mL) and the procedure was repeated. Fmoc deprotection was performed by treating the resin with 20% piperidine in DMF for 10 min (×2), followed by washing with DCM (5 × 2 mL) and DMF (5 × 2 mL). For a 10 μmols scale synthesis on TGA-NovaSyn resin (loading−0.25 mmol/g), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (HATU, 40 μmols, 15.2 mg), hydroxybenzotrilazole (HOBT, 40 μmols, 5.4 mg), diisopropylethylamine (DIPEA, 80 μmols, 14 µL), and Fmoc-amino acids/Fmoc-PNA monomers (40 μmols) were mixed in dry DMF (0.4 mL). After 5 min of pre-activation, the solution was transferred to the resin. After 60 min, the reaction mixture was discarded, and the resin was washed with DCM (5 × 2 mL) and DMF (5 × 2 mL). The PNA−peptide conjugates were deprotected and released from the resin by treatment with 90:10 (v/v) TFA/m-cresol for 2 h (2 × 1 mL). The PNAs were triturated with cold diethyl ether, and the precipitate was collected by centrifugation and decantation of the supernatant. The residues were dissolved in water and purified by semi preparative HPLC using a Dionex UltiMate 3000 HPLC system (ThermoFisher Scientific, Waltham, MA, USA) with automatic fraction collection. A semi-preparative C18 reversed-phase column (Jupiter C18, 10u, 300Å, 250 × 10 mm, Phenomenex) was used with a linear gradient of eluents A (0.1% TFA in water) and B (MeCN) at a flow rate of 4 mL/min. Mass analysis of FIT-PNAs was acquired by MALDI-TOF MS (Bruker Daltonics, Microflex LRF) using 2,5-Dihydroxybenzoic acid (DHB) as a matrix.

The melting temperatures (T_m) of the PNA: RNA/DNA duplexes were determined using UV melting curves recorded on an Evolution One Plus UV-Vis Spectrophotometer. Solutions of the FIT-PNAs and their complementary RNAs (1:1 ratio) were prepared in a PBS buffer (100 mM NaCl, 10 mM NaH₂PO₄, pH 7) and adjusted to a final duplex concentration of 2 µM. Prior to analysis, the samples were heated from 20 °C to 90 °C at a rate of 5 °C/min and then cooled back to the starting temperature at a rate of 2 °C/min. Absorbance at 260 nm was monitored as the temperature increased to 90 °C at a rate of 1 °C/min. Each measurement was repeated at least twice, with the T_m value representing the average value of the inflection point.

Fluorescence spectra were recorded by using a Jasco FT-6500 spectrometer. Measurements were carried out in fluorescence quartz cuvettes (10 mm). Solution of the FIT-PNA and the RNA/DNA (ratio 1:2) were prepared in a PBS buffer (pH 7.0) at 37 °C for 2 h.

UV-Vis spectra of CCAT1 FIT-PNAs were recorded using an Evolution One Plus UV-Vis Spectrophotometer. FIT-PNA solutions, either with or without the presence of RNA synthetic RNA, were prepared in PBS buffer (100 mM NaCl, 10 mM NaH₂PO₄, pH 7). Prior to measurement, the FIT-PNA:RNA duplex solutions were annealed at 37 °C for 2 h. Full spectrum was recorded in the range of 200–800 nm.

CD spectra were acquired using a Jasco F-1100 spectropolarimeter equipped with a temperature-controlled sample holder. Samples included both single-stranded FIT-PNA and FIT-PNA:RNA duplexes (1:1 M ratio), prepared at a final FIT-PNA concentration of 15 µM in PBS buffer (100 mM NaCl, 10 mM NaH₂PO₄, pH 7.0). Hybridization was carried out by incubating the samples at 37 °C for 2 h. CD measurements were performed at 25 °C using a 1 mm pathlength quartz cuvette with a total volume of 200 µL. Spectra were recorded over 200–320 nm range, and each final spectrum represents an average of five replicates.

Quantum yields for all FIT-PNAs were calculated using Cresyl Violet as a reference fluorescent dye [62–64]. Each FIT-PNA (4, 6 and 8 µM) was hybridized to complementary and G-mismatched RNA in PBS (pH 7.0) at a 1:2 ratio, respectively, and incubated at 37 °C for 2 h. The samples were excited at 580 nm, and emission spectra were recorded between 400 and 750 nm.

Limit of detection (LOD) of all FIT-PNAs was recorded by using a Cytation 3 plate reader. Measurements were carried out in Greiner 96 well black plates with flat bottom in a Tris-EDTA buffered solution (25 mM Tris-EDTA, 150 mM NaCl with 0.05% Tween-20). The FIT-PNA’s concentration was constant in the duplex solution (0.5 µM) while the RNA was added in different concentrations. All the FIT-PNAs were incubated with the complementary RNA at 37 °C for 2 h on the plate for annealing. LOD was calculated according to the formula: LOD = 3.3*σ/slope [65].

Total RNA from the cells was isolated using TRIzol reagent (ThermoFisher Scientific, Waltham, USA) following the manufacturer’s instructions and quantified using a NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific, Waltham, USA). Reverse transcription of RNA(1 µg) into cDNA was performed using the QScript cDNA Synthesis Kit (Quantabio, Beverly, MA, USA) according to the manufacturer’s instructions. RT-qPCR was conducted to on a CFX Connect Real-Time PCR Detection System (BioRad, Hercules, CA, USA) using PerfeCTa SYBER® Green FastMix qPCR reagent (Quantabio, Beverly, MA, USA). The primers used are described in Supplementary Material (Supplementary Table S4), they were purchased form IDT (Coralville, USA) and HyLabs (Rehovot, Israel). The target genes were amplified under the following thermocycling conditions: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The specificity of the PCR products was verified by analyzing the melting curves. The relative expression of target genes was calculated using the 2^−ΔΔCT method, and expression levels were normalized to the housekeeping gene Ribosomal protein lateral stalk subunit P0 (RPLP0).

OVCA433 and SKOV3 cells were grown in EMEM and McCoy’s 5A, respectively, (Beit Haemek Biological Industries, Israel) supplemented with 10% (v/v) FBS, 100 U/mL penicillin; 0.1 mg/mL streptomycin; 2 mM L-Glutamine, at 37 °C with 5% CO₂. Cells were routinely checked for mycoplasma contamination using MycoBlue Mycoplasma detector Kit (Vazyme, China).

FACS analysis of FIT-PNA uptake was conducted by seeding OVCA433 (50 × 10⁴) and SKOV3 (35 × 10⁴) cells into 6-well plates, allowing them to adhere overnight under standard culture conditions until they reached 70%–80% confluence. The medium was replaced, and the cells were incubated with 2 µM FIT-PNAs at 37 °C in a humidified atmosphere containing 5% CO₂ for 5 h. Following thorough washing, the cells were harvested using 0.25% Trypsin-EDTA (3 min at 37 °C), collected into 15 mL Falcon tubes, and centrifuged at 1,200 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 350 μL cold PBS, which was then filtered through 70 µm Falcon Cell Strainers. The samples were analyzed using a Fortessa FACS analyzer (Core Research Facilities, The Hebrew University of Jerusalem, Jerusalem, Israel). The cells were gated based on normalized fluorescence of untreated cells to determine the percentage of cells that internalized the FIT-PNAs. Data analysis was performed using FlowJo 10.10 software.

FACS data are presented as the mean ± SD from experiments. At least two independent experiments were performed per assay, each with Two technical replicates. Statistical significance was determined using a One-way or Two-way ANOVA test with P < 0.001 considered extremely significant (***), P < 0.01 highly significant (**), and P < 0.05 statistically significant (*). mRNA expression, as measured by RT-qPCR, was normalized to the control cell expression, and the data represent the average of two biological replicates, each with corresponding duplicates. Statistical analysis was carried out using Student’s t-test, with P < 0.05 considered statistically significant (*).

Twenty-four hours prior to PNA addition, OVCA433 cells (60 × 10³) and SKOV3 cells (50 × 10³) were seeded onto µ-slide 8-well chambers (ibidi GmbH, Gräfelfing, Germany) and incubated at 37 °C with 5% CO₂ until reaching 60%–70% confluence. The cells were rinsed with 1× PBS and treated with 2 µM FIT-PNAs in medium at 37 °C for 5 h. After incubation, the cells were washed twice with 1× PBS and stained with Hoechst (1 μg/mL) for 15 min at room temperature. The cells were then washed again with 1× PBS, and 300 µL of 1× PBS was added to each well for live cell observation. Control cells included OVCA433 and SKOV3 cells that were not treated with FIT-PNA. Cell fluorescence observations were performed using a Nikon AIR+ confocal microscope (Core Research Facilities, The Hebrew University of Jerusalem, Israel) and images were analyzed using NIS-Elements AR software (version 5.21).

Double-stranded PNA molecules were constructed using the Proto Nucleic Acid Builder (pNAB) software, where the 5′ to 3′ sequence of the target RNA/DNA strand was used as the N- to C-terminal input for PNA strand generation. The resulting PNA:PNA duplex structures were analyzed using the x3DNA server to obtain helical parameters. The generated parameter file was manually edited (replacing “T” with “U”) and used to model corresponding RNA:RNA and B-form DNA:DNA duplexes using x3DNA. Relevant single strands from the pNAB-generated PNA:PNA duplex and the x3DNA-generated RNA:RNA or DNA:DNA duplex were extracted and saved as individual PDB files. These were then docked into PNA:RNA and PNA:DNA duplexes using the HNADOCK server. The resulting duplex structures were processed in Schrödinger Maestro (v.14.0) for structure preparation. Final structures were energy-minimized using the OPLS4 force field implemented in Maestro. Further details are provided in the Supplementary Material.

Based on the simple one-step synthetic of G⁺ PNA monomer [51], we prepared in quantitative yields the cpG⁺ PNA monomer starting from the Fmoc-protected cpG monomer [60] (Scheme 1). The final product was used after a simple workup and was fully characterized by NMR and HRMS (Supplementary Figures S35, S36).

In this study, we have synthesized a series of G-modified FIT-PNAs (Table 1) that target the lncRNA CCAT1. This RNA biomarker has been previously studied in our lab for FIT-PNA based diagnosis in colorectal cancer where CCAT1 was detected in unfixed cancer cell lines [18] and in fresh human cancer tissues [23]. The choice of the BisQ surrogate base (Scheme 2) was for several reasons: (1) ease of synthesis [61]; (2) superior RNA sensing in comparison to the TO surrogate base [61]; and (3) red-shifted emission (λ_em,max = 613 nm) that is more suitable for biological samples (lower background fluorescence from biological samples). Three types of G modifications adjacent to BisQ were installed: G⁺, cpG, and cpG⁺. FIT-PNAs were synthesized on the solid support (Novasyn TGA resin) using standard Fmoc-based peptide/PNA Chemistry. To provide water solubility and cellular uptake, FIT-PNAs were installed with a short peptide ((D)K₄) that has higher stability in biological medium than the L-peptide (K₄), as previously reported [2]. After FIT-PNA cleavage from the solid support, the FIT-PNA oligomers were purified by HPLC and analyzed by MALDI-TOF MS (Supplementary Figures S1–S4).

FIT-PNAs were annealed to a fully complementary 15-mer RNA, and the fluorescence of the duplexes was measured (Figure 1). Among the sequences tested, cpG⁺ FIT-PNA exhibited the most pronounced response, showing over a twofold increase in fluorescence compared to the unmodified G FIT-PNA. While single modifications on G (G⁺ and cpG) also enhanced fluorescence, their performance was less effective than the double modification. Overall, the data demonstrate that the combined chemical modifications on G synergistically improve the fluorescence response, making cpG⁺ FIT-PNA the most responsive RNA probe.

We next explored the sequence selectivity of FIT-PNAs by measuring the fluorescence of FIT-PNAs with RNA sequences that have a single mismatch at the nucleobase opposite to the modified G base in the FIT-PNA sequence. To our surprise, we found higher emission for all FIT-PNA sequences for the GG mismatch in RNA (Figure 3A; Supplementary Figure S16). This was not the case for a GG mismatch in DNA (Figure 3C). All other mismatches were well-discriminated by FIT-PNAs (Supplementary Figure S16 for RNA; Supplementary Figure S17 for DNA).

The overall photophysical properties of FIT-PNAs were assessed by measuring three key parameters: brightness (BR = QY × ε_max), fluorescence increases upon RNA hybridization (I/I₀), and quantum yield (QY) (Table 2). These measurements also included the GG mismatch RNA for all FIT-PNAs.

As shown in Table 2, G⁺ FIT-PNA exhibited parameters similar to those of the unmodified G FIT-PNA, indicating minimal improvement in photophysical performance. For example, QY values were 0.11 and 0.13 for fully matched (FM) and GG mismatch RNA, respectively, comparable to 0.09 and 0.11 for G FIT-PNA. In contrast, cpG FIT-PNA demonstrated increased responsiveness, with QYs of 0.17 and 0.24 for FM and GG mismatch RNA. Most notably, the cpG⁺ FIT-PNA achieved the best results, with approximately a threefold increase in both QY and brightness (QY = 0.29; BR = 27.8) with GG mismatch RNA compared to G FIT-PNA. Its fluorescence enhancement over background in the single-stranded form was also significant, with I/I₀ values of 10.5 and 14.3 for FM and GG mismatch RNA, respectively.

Moreover, a slight increase in absorbance values was observed in the BisQ absorbance region (λ_max,abs= ∼590 nm) for the duplex formed with G_mm RNA compared to the fully matched RNA duplex (Figure 2B; Supplementary Figures S18–S20). This was observed for all FIT-PANs (modified and unmodified).

Limit of detection (LOD) is defined as the lowest concentration of RNA detected by a particular probe. All modified FIT-PNAs exhibited a lower LOD compared to the unmodified G FIT-PNA (Supplementary Figure S26; Table 2). Specifically, the LOD values for fully matched RNA decreased from 5.00 nM for the unmodified probe to 2.67, 2.00, and 1.56 nM for G⁺, cpG, and cpG⁺ FIT-PNAs, respectively. cpG⁺ FIT-PNA also showed the lowest LOD with G_mm RNA (2.6 nM). However, all FIT-PNAs exhibited higher values for the LODs (inferior) for the G_mm RNA compared to the fully matched RNA.

We also measured melting temperatures (T_m) for FIT-PNAs with FM and GG mismatch RNA (Table 2; Supplementary Figures S8–S11). For FM RNA, the presence of a positive charge on G (G⁺) generally decreased duplex stability, shown by a T_m reduction of about 3 °C for G⁺/G and around 4.9 °C for cpG⁺/cpG. Conversely, the cpG modification increased T_m value only for cpG FIT-PNA. All FIT-PNAs exhibited lower T_m values with GG mismatch RNA, indicating decreased duplex stability. Interestingly, there was an inverse correlation: the lower stability of the GG mismatch duplex corresponded with higher fluorescence intensity across all FIT-PNAs. FIT-PNAs were also tested with synthetic DNA, where no fluorescence increase was observed for GG mismatches, and all mismatches were well-resolved (Supplementary Figure S17; Supplementary Table S3), in accordance with molecular simulations (Figures 3C,D, 5).

CD spectroscopy was also performed on all FIT-PNA sequences in the presence and absence of complementary RNA and G_mm RNA (Figure 2A; Supplementary Figure S21) to investigate molecular interactions and assess the structural stability of the formed duplexes. As expected for single-stranded FIT-PNAs, including the cpG and cpG⁺ modified variants, no detectable CD signals were observed [66]. Upon hybridization, both FIT-PNA:RNA and FIT-PNA:G_mm RNA duplexes exhibited characteristic CD signatures of antiparallel PNA:RNA heteroduplexes [3, 67]. The CD signals of the FIT-PNA:G_mm RNA duplexes were less intense in the ∼260–270 nm region, and a slight spectral shift was observed, suggesting altered helical organization and reduced duplex stability. Nonetheless, both duplex types displayed a similar maximum at ∼210–220 nm and a minimum at ∼240–245 nm. These observations align with the thermal melting (T_m) data, where FIT-PNAs showed lower T_m values when hybridized to G_mm RNA compared to the fully matched RNA.

To provide some insight into these observations, we modelled the structures of “fully matched BisQ FIT-PNA:RNA duplex,” “G-G mismatched BisQ FIT-PNA:RNA duplex,” “fully matched BisQ FIT-PNA:DNA duplex,” and “G-G mismatched BisQ FIT-PNA:DNA duplex” (detailed in ESI, Supplementary Figures S37–S41). It is noteworthy that these molecular simulations were done at the ground state of these molecules.

Ten ns stochastic dynamics simulations were conducted to monitor the dihedral angle (ω) between the two quinoline rings of BisQ (Scheme 2) and their π-π stacking interactions with neighboring nucleobases.

We observed that in the fully matched duplex (with RNA), ω mainly ranged from −100° to −140°, with dominantly the inner quinoline ring π-stacking effectively, and the outer ring exhibited weak or no π-stacking at all. In contrast, the G-G mismatched duplex (with RNA), predominantly showed ω between −40° and −80°, with both rings forming face-to-face π-π interactions. This may explain the higher fluorescence observed for a G-G mismatch in RNA. Analysis of dihedral angle populations (ω) during 10 ns shows a clear difference between the duplex types (Figures 3B,D). For the G-G mismatch with RNA, more population density lies between −40° and −80°, favorable for π–π stacking of both quinoline rings (Figure 3B, blue trace). In the fully matched duplex (with RNA), ω predominantly falls between −100° and −140°, a range unsuitable for stacking of the outer quinoline ring (Figure 3B, black trace). For DNA, the population densities are strikingly different (Figure 3D). For the G-G mismatch with DNA, ω spreads all over (Figure 3D, red trace) with no distinct population density at the −40° to −80° range. With FM (with DNA), there is a distinct population at this range (Figure 3D, black trace), albeit lower than that of G-G mismatch with RNA (Figure 3B, blue trace). Altogether, the results shown in Figures 3B,D correlate with the spectroscopic data (Figures 3A,C).

To validate our observation that the value of ω ranging from −40° to −80° is suitable for π−stacking of both quinoline rings in BisQ, we further modelled and simulated a total of 4 FIT-PNAs that include X-BisQ in each probe (where X = A, G, C, or T, Supplementary Figures S1, S5–S7), and performed a correlation study between the percentage of well-stacked population of BisQ (where both quinoline rings are stacked between neighboring bases) and the percentage of population where ω ranges from −40° to −80° over 10 ns (Supplementary Figures S41a). We obtained a 0.82 Pearson’s correlation coefficient (Supplementary Figures S41b). Subsequently, we validated this correlation to the experimental value of fluorescence against π-stacked BisQ population which provided a Pearson’s correlation coefficient of ca. 0.74 (Supplementary Figures S41c). Statistically, these two values suggest a good correlation between these parameters (fluorescence and π-stacked BisQ population).

We next studied the dynamics of ω over each nanosecond (for 6 ns, data is shown in Figures 4, 5), focusing on either poor π-stacking (−100° < ω < −140°) or appreciable π-stacking (−40° < ω < −80°). For RNA, ω is quite stable for both FM and GG mismatch (Figure 4A). The well-stacked structures of BisQ for GG mismatch consist of 60%–80% of all structures generated during this timeframe (Figure 4B, blue trace). In contrast, for FM RNA, this value drops down to 1%–10% (Figure 4B, black trace).

For DNA, ω is much more dynamic in this timeframe (Figure 5A, 6 ns). For FM BisQ FIT-PNA:DNA duplex (Figure 5A, black boxes for each 1 ns of simulation), the outer quinoline ring of BisQ is initially well stacked in the duplex (ca. 80% of all structures during the first 2 ns) but gradually drops to ca. 30% after 4 ns (Figure 5B, black trace). In contrast, the well-stacked structures for GG mismatch DNA (Figure 5A, red boxes for each 1 ns of simulation) consist of only ca. 30% and decrease to ca. 18% during the remaining 5 ns of the simulation (Figure 5B red trace).

To improve water solubility and cellular uptake, FIT-PNAs were conjugated to a short positively charged peptide (4 D-Lysines, (D)K₄) at the C-terminus. We studied their ability to track lncRNA CCAT1 in two ovarian cancer cell lines: SKOV3, which expresses high levels of CCAT1 (confirmed by RT-qPCR), and OVCA433, which has minimal CCAT1 expression (Supplementary Figure S27; Supplementary Table S4). Cells were treated with 2 µM of modified and unmodified FIT-PNAs for 3 h at 37 °C, and fluorescence was analyzed via flow cytometry (Figure 6). All FIT-PNAs showed higher fluorescence in SKOV3 than in OVCA433. Notably, cpG⁺ FIT-PNA produced the strongest signal in both cell lines, with an approximately 8-fold higher fluorescence in SKOV3. It was the only modified probe that outperformed the unmodified FIT-PNA in both cell types (Supplementary Figure S28). In contrast, G⁺ and cpG FIT-PNAs showed no significant difference from the unmodified probe, indicating that these modifications offered no added benefit when adjacent to BisQ.

Live-cell imaging (Figure 7; Supplementary Figures S33–S34) supported these findings: SKOV3 and OVCA433 cells incubated with 2 µM FIT-PNAs for 5 h and stained with Hoechst showed higher fluorescence for cpG⁺ FIT-PNA in SKOV3. In OVCA433, only minimal fluorescence was observed for cpG⁺ FIT-PNA, and signals from other probes were undetectable. Overall, cpG⁺ FIT-PNA was the most effective for RNA detection in SKOV3 cells, with fluorescence levels correlating with CCAT1 expression, demonstrating its specificity and potential as a targeted probe for ovarian cancer cells.

RNA sensing molecules have been developed for a variety of biomedical indications such as identifying RNA biomarkers related to disease.

Chemically modified FIT-PNAs are shown to improve the biophysical properties of these RNA sensors.