Abstract RNA and DNA biomarkers serve as diagnostic molecules for detecting a specific disease by a variety of methods and technologies. A common approach is based on a fluorescence readout signal generated after hybridization to a target nucleic acid sequence. One such technology is termed Forced-Intercalation Peptide Nucleic Acid (FIT-PNA). In the FIT-PNA design, one of the nucleobases of the PNA sequence is replaced by a fluorescent molecule termed as “surrogate base.” One of the more common fluorophores explored to date is the cyanine dye, Thiazole Orange (TO). In this report, we have designed TO-based FIT-PNAs that are chemically modified with a cyclopentane backbone (cpTO). In addition, we have introduced to the FIT-PNA design either a cyclopentane T (cpT) or a tetrahydrofuran T (THFT) flanking cpTO. In a model system (11-mer FIT-PNA), we observe a dramatic increase in fluorescence (with DNA or RNA complementary sequences) for both cpT-cpTO and THFT-cpTO FIT-PNAs in comparison to the unmodified counterpart (T-TO FIT-PNA). Moreover, sequence specificity for an RNA sequence with a single mismatch is dramatically improved for both cpT-cpTO and THFT-cpTO FIT-PNAs. Molecular simulations of both cpT-cpTO FIT-PNA and TO (unmodified) FIT-PNA further support the superiority of these chemically modified nucleic-acid probes, as corroborated by a greater π–π stacking of cpTO in the PNA-RNA duplex. Lastly, a cpT-cpTO FIT-PNA targeting the oncogenic long noncoding RNA ANRIL (antisense noncoding RNA in the INK4 locus) was shown to detect this RNA biomarker in ovarian cancer cells (OVCAR-8). This probe was superior to the unmodified TO-based FIT-PNA, highlighting the added value of chemically modified TO FIT-PNAs as means for obtaining highly sensitive and sequence-specific nucleic acid sensors.

A variety of oligonucleotide-based probes have been developed for specific and selective sensing of RNA and DNA. Among these, FIT-PNAs (forced intercalation-peptide nucleic acids) and FIT probes (DNA- and RNA-based sensors) have been studied for a variety of RNA biomarkers in cell culture and tissues, and in vivo. FIT-PNAs and FIT probes are RNA/DNA sensors that exhibit fluorescence upon sequence-specific RNA/DNA hybridization. Several synthetic approaches have been successfully applied to increase the brightness and selectivity of these molecules, including the introduction of cyclopentane (cp) modified PNA monomers (cpPNA) as well as locked nucleic acids (LNAs-for FIT probes). In this report, we have explored the biophysical properties of FIT-PNAs that are modified with gamma-l-serine PNAs (γPNAs). We found that introducing a single γ-PNA flanking the fluorophore (BisQ) in the FIT-PNA sequence is sufficient to achieve a 46-fold increase in fluorescence for the PNA:RNA duplex, similarly to cpPNA. Interestingly, when two γ-PNAs flank BisQ on both sides, a significant increase in RNA affinity is observed (over an 8 °C increase in melting temperature, T m). Altogether, γ-PNAs are a beneficial chemical modification that leads to brighter FIT-PNAs with improved binding affinities to targeted RNA.

Peptide nucleic acid (PNA) is a synthetic DNA analogue characterized by exceptional biostability and strong hybridization affinity toward complementary DNA and RNA. However, its inherently low membrane permeability hampers its biomedical applicability. N-Methylation of guanine and adenine PNA monomers produces a positively charged nucleobase, which suppresses the formation of self-duplexes while improving DNA affinity through electrostatic interactions. To overcome the cellular delivery limitations of PNA, we designed and synthesized a 16-mer, nontargeted model PNA incorporating 4, 6, or 8 positively charged purines (G+ and A+). As comparative controls, the corresponding unmodified PNA was conjugated to a short cell-penetrating peptide (CPP) containing 4, 6, or 8 d-lysine residues. All constructs were labeled with Rhodamine B to enable quantitative cellular uptake analysis. Flow cytometry and confocal microscopy in OVCAR-8 ovarian cancer cells revealed that the PNA incorporating six positively charged purines (MCP6, multiple charged purines with an overall 6 positive charges) exhibited markedly enhanced cellular internalization compared to both the other MCP-PNAs and the CPP-PNA controls. MCP-PNAs showed no noticeable signs of cell toxicity, and their binding affinities (thermal melting profiles) were comparable to CPP-PNAs. In addition, MCP-PNAs well discriminated single mismatches in RNA, similarly to CPP-PNAs. Overall, this strategy provides a simple and effective approach for generating inherently cell-permeable PNAs.

FIT-PNAs (forced intercalation-Peptide Nucleic Acids) are promising RNA sensors due to the enhanced fluorescence gained by such molecules upon RNA hybridization. In this report we describe a chemical approach that leads to unprecedented brightness for a FIT-PNA where the neighbouring Guanine base (G) to the fluorophore (a.k.a. surrogate base) is chemically modified with a cyclopentane (cp) backbone and is N-methylated, leading to a positively charged (G+) base. A series of G modified bases (G+, cpG, and cpG+) were introduced as the neighbouring base to BisQ (surrogate base) in 15-mer FIT-PNAs designed to sense the oncogenic long-noncoding RNA, colon cancer associated transcript 1 (lncRNA CCTA-1). Using synthetic RNA, the combination denoted as cpG+ led to a two-fold increase in brightness (BR = 16.9) compared to the unmodified G base (BR = 8.4). Introducing a G mismatch in RNA sequence that is opposite to the G base (G, G+, cpG, or cpG+) in the FIT-PNA, led to an increase in fluorescence that was not observed for synthetic DNA. Molecular simulations confirmed these observations and further correlated fluorescence data for FIT-PNAs with synthetic DNA and RNA with/out mismatches. Importantly, in ovarian cancer cells overexpressing CCAT1, only the cpG+ modified FIT-PNA produced a bright fluorescent signal, confirmed by FACS and confocal microscopy. Our results demonstrate that strategic chemical modifications of the neighboring G base in FIT-PNA significantly enhance their brightness and specificity for RNA detection in biological systems.

Peptide nucleic acids (PNA) [1] are synthetic DNA analogs that are used as DNA/RNA mimics for diagnostic [2, 3] and therapeutic purposes [2-5]. One sensing strategy is the use of fluorogenic probes. One such probe is forced intercalation (FIT) PNA, which has a surrogate base as a cyanine dye (e.g., BisQ, TO) that replaces one of the natural bases in the PNA sequence. FIT-PNAs fluoresce after DNA/RNA hybridization, and display sensitivity towards single base mismatches [6]. These, combined with other desirable features, render them highly attractive as RNA/DNA sensors.Herein, we describe the procedure for FIT-PNA synthesis, both by manual and automated methodologies.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

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.

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.

Correction for 'Cyclopentane FIT-PNAs: bright RNA sensors' by Odelia Tepper et al., Chem. Commun., 2021, 57, 540-543, https://doi.org/10.1039/D0CC07400D.

The aim of this paper was to evaluate the effect of vehicle, chemical enhancer and iontophoresis on the skin accumulation of clobetasol propionate (CP) and mometasone furoate (MF). In vitro permeation experiments were performed using pig ear skin as barrier and HPLC as quantification method. The formulations tested were chitosan gels, sodium-deoxycholate gels and commercial creams of CP and MF. The results obtained indicate that Na-DOC gel had an enhancing effect on the skin accumulation of both active agents. This effect was more evident with CP especially in the stratum corneum and epidermis which are the target sites of topical steroidal treatment. Two terpene derivatives (d-limonene and nerolidol) and Transcutol® P were evaluated as chemical penetration enhancers. Nerolidol produced considerable increase in the amount of CP and MF accumulated without any permeation across the skin. The application of electric current (anodal iontophoresis) to the gels improved the accumulation of MF while it did not effect the accumulation of CP. Due to the best accumulation results of nerolidol, the enhancement effect in combination with iontophoresis was also investigated. It was shown that, the combination of anodal iontophoresis and chemical enhancer (nerolidol) produced no further enhancement for both active agents.

The DNA mimic, PNA (peptide nucleic acid), has been with us now for almost 3 decades [...]

Cyclopentane modified FIT-PNA (cpFIT-PNA) probes are reported as highly emissive RNA sensors with the highest reported brightness for FIT-PNAs. Compared to FIT-PNAs, cpFIT-PNAs have improved mismatch discrimination for several pyrimidine-pyrimidine single nucleotide variants (SNVs).

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.

Osteosarcoma (OS) is an aggressive malignancy affecting mostly children and adolescents. MicroRNAs (miRNAs) play important roles in OS development and progression. Here we found that miR‐16‐1‐3p and miR‐16‐2‐3p “passenger” strands, as well as the “lead” miR‐16‐5p strand, are frequently downregulated and possess strong tumor suppressive functions in human OS. Furthermore, we report different although strongly overlapping functions for miR‐16‐1‐3p and miR‐16‐2‐3p in OS cells. Ectopic expression of these miRNAs affected primary tumor growth, metastasis seeding and chemoresistance and invasiveness of human OS cells. Loss‐of‐function experiments verified tumor suppressive functions of these miRNAs at endogenous levels of expression. Using RNA immunoprecipitation (RIP) assays, we identify direct targets of miR‐16‐1‐3p and miR‐16‐2‐3p in OS cells. Moreover, validation experiments identified FGFR2 as a direct target for miR‐16‐1‐3p and miR‐16‐2‐3p. Overall, our findings underscore the importance of passenger strand miRNAs, at least some, in osteosarcomagenesis.

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 (C9-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 C9-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 C9-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.

To date, there are limited approaches for the direct and rapid visualization (on site) of tumor tissues for pathological assessment and for aiding cytoreductive surgery. Herein, we have designed FIT-PNAs (forced-intercalation-peptide nucleic acids) to detect two RNA cancer biomarkers. Firstly, a lncRNA (long noncoding RNA) termed CCAT1, has been shown as an oncogenic lncRNA over-expressed in a variety of cancers. The latter, an mRNA termed KRT20, has been shown to be over-expressed in metastases originating from colorectal cancer (CRC). To these FIT-PNAs, we have introduced the bis-quinoline (BisQ) cyanine dye that emits light in the red region (605-610 nm) of the visible spectrum. Most strikingly, spraying fresh human tissue taken from patients during cytoreductive surgery for peritoneal metastasis of colon cancer with an aqueous solution of CCAT1 FIT-PNA results in bright fluorescence in a matter of minutes. In fresh healthy tissue (from bariatric surgeries), no appreciable fluorescence is detected. In addition, a non-targeted FIT-PNA shows no fluorescent signal after spraying this FIT-PNA on fresh tumor tissue emphasizing the specificity of these molecular sensors. This study is the first to show on-site direct and immediate visualization of an RNA cancer biomarker on fresh human cancer tissues by topical application (spraying) of a molecular sensor.

Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent‐accessible residues. Here we show that the disordered C‐terminal domain (CTD) of HIV‐1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.

The gene encoding the kinase Mnk2 (MKNK2) is alternatively spliced to produce two isoforms-Mnk2a and Mnk2b. We previously showed that Mnk2a is downregulated in several types of cancer and acts as a tumor suppressor by activation of the p38-MAPK stress pathway, inducing apoptosis. Moreover, Mnk2a overexpression suppressed Ras-induced transformation in culture and in vivo. In contrast, the Mnk2b isoform acts as a pro-oncogenic factor. In this study, we designed modified-RNA antisense oligonucleotides and screened for those that specifically induce a strong switch in alternative splicing of the MKNK2 gene (splice switching oligonucleotides or SSOs), elevating the tumor suppressive isoform Mnk2a at the expense of the pro-oncogenic isoform Mnk2b. Induction of Mnk2a by SSOs in glioblastoma cells activated the p38-MAPK pathway, inhibited the oncogenic properties of the cells, re-sensitized the cells to chemotherapy and inhibited glioblastoma development in vivo. Moreover, inhibition of p38-MAPK partially rescued glioblastoma cells suggesting that most of the anti-oncogenic activity of the SSO is mediated by activation of this pathway. These results suggest that manipulation of MKNK2 alternative splicing by SSOs is a novel approach to inhibit glioblastoma tumorigenesis.