A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

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.

Correction: Cyclopentane FIT-PNAs: bright RNA sensors.

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.

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

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 (dK8), 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.

Cyclopentane FIT-PNAs: bright RNA sensors.

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).

Peptide Nucleic Acids: Applications in Biomedical Sciences.

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

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

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.

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

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.

MiR-16-1-3p and miR-16-2-3p possess strong tumor suppressive and antimetastatic properties in osteosarcoma.

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.

Specific inhibition of splicing factor activity by decoy RNA oligonucleotides.

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.

Modulation of MKNK2 alternative splicing by splice-switching oligonucleotides as a novel approach for glioblastoma treatment.

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.

Predictive Model for the Sequence-Dependent Fluorogenic Response of Forced-Intercalation Peptide Nucleic Acid.

The forced-intercalation peptide nucleic acid (FIT-PNA) concept, introduced by Seitz and co-workers, is based on replacing a nucleobase of the PNA sequence with a cyanine dye (such as thiazole orange). The cyanine dye is thus a surrogate base that is forced to intercalate in the duplex (e.g. , PNA: DNA). This allows single-mismatch sensitivity as the introduction of a mismatch in the vicinity of the dye increases freedom of motion and leads to a significant depletion of its fluorescence because of the free rotation of the monomethine bond separating the two π-systems of the cyanine dye. Herein, we designed and synthesized six FIT-PNA probes, featuring bisquinoline (BisQ), a red-emitting cyanine dye recently developed in our laboratory for FIT-PNAs. By following PNA-DNA duplex fluorescence, we found new sequence-based factors governing the fluorescence response to the mismatched FIT-PNA:DNA duplex. Fluorogenic properties are correlated with the π-stacking energy of three distinctive base pair steps (BPSs) in the PNA:DNA duplex. The first two are the two BPSs opposite BisQ, whereas the third is the BPS of the mismatch position, which presumably becomes unstacked due to the mismatch. We suggest a predictive model for FIT-PNA single-mismatch detection mechanism, a model that can be used in future research to improve FIT-PNA design.

Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of the combination therapy to mice bearing intracranial murine glioblastoma resulted in marginal therapeutic outcomes, probably due to brain delivery challenges, p53 mutation status, and the aggressive nature of the selected cell line. Nevertheless, this study provides a proof of concept for exploiting regulators of tumor dormancy for glioblastoma therapy. This therapeutic strategy can be exploited for future investigations using a variety of therapeutic entities that manipulate the expression of dormancy-associated genes in glioblastoma as well as in other cancer types.-Tiram, G., Ferber, S., Ofek, P., Eldar-Boock, A., Ben-Shushan, D., Yeini, E., Krivitsky, A., Blatt, R., Almog, N., Henkin, J., Amsalem, O., Yavin, E., Cohen, G., Lazarovici, P., Lee, J. S., Ruppin, E., Milyavsky, M., Grossman, R., Ram, Z., Calderón, M., Haag, R., Satchi-Fainaro, R. Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Protein Regulation by Intrinsically Disordered Regions: A Role for Subdomains in the IDR of the HIV-1 Rev Protein.

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.

CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice Switching Oligonucleotides.

Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA-peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.

The effect of linker type and recognition peptide conjugation chemistry on tissue affinity and cytotoxicity of charged polyacrylamide.

To increase colonoscopy competence in ambiguous situations (e.g. the existence of flat polyps), an explicit in situ (at real time) diagnosis at the molecular level is required. We have previously shown that the affinity of fluorescent cationic polyacrylamide (Flu-CPAA) to malignant regions in the colon mucosa can be improved by conjugating the recognition peptide EPPT1 to the polymer backbone (to form Flu-CPAA-Pep). Using another recognition peptide, namely VRPMPLQ, we elucidated in the present study the effect of linker type and conjugating methods on Flu-CPAA-VRPMPLQ cytotoxicity and on its affinity to cell lines as well as human colorectal cancer (CRC) biopsies. In order to derive the relationship between the response variable and the experimental factors in a minimal set of experiments, a computerized statistical design of experiment (DoE) strategy was implemented. Data were collected in a six-factor factorial design to study the effect of experimental factors (independent variables) on the ability of the Flu-CPAA polymers to bind specifically to the colon cancer cell lines or the human biopsies (the response). It was found that the presence of VRPMPLQ on the Flu-CPAA improved the polymer's affinity to the human CRC biopsies and to the colon cancer cell lines representing stage B in the Duke severity staging system. The cytotoxicity of Flu-CPAA with high charge density was reduced after conjugated with VRPMPLQ. The replacement of Ahx linker by PEG linker of similar length did not affect the affinity to the human biopsies, nor did it affect cytotoxicity. However, elongating the PEG linker reduced the in vitro affinity to the colon cancer cell lines and to human CRC biopsies. Changing the conjugation method from condensation (amide bond formation) to the click conjugation method did not affect the affinity properties of the polymers. It did reduce, however, the polymer cytotoxicity. We suggest that Flu-CPAA-Pep, with the VRPMPLQ peptide as a recognition moiety, could serve for early diagnosis and screening of CRC patients during endoscopic procedures.

Solid nano-in-nanoparticles for potential delivery of siRNA.

siRNA-based therapeutics possess great potential to treat a wide variety of genetic disorders. However, they suffer from low cellular uptake and short half-lives in blood circulation; issues that remain to be addressed. This work is, to the best of our knowledge, the first to report the production of solid nano-in-nanoparticles, termed double nano carriers (DNCs) by means of the innovative technology of nano spray drying. DNCs (with a median size of 580-770nm) were produced by spraying at low temperatures (50°C) to prevent damage to heat-sensitive biomacromolecules like siRNA. DNCs consisting of Poly (d,l-lactide-co-glycolide) used as a wall material, encapsulating 20% human serum albumin primary nanoparticles (PNPs) loaded with siRNA, were obtained as a dry nanoparticulate powder with smooth spherical surfaces and a unique inner morphology. Incubation of pegylated or non-pegylated DNCs under sink conditions at 37°C, elicited a controlled release profile of the siRNA for up to 12 or 24h, respectively, with a minimal burst effect. Prolonged incubation of pegylated DNCs loaded with active siRNA (anti EGFR) in an A549 epithelial cell culture monolayer did not induce any apparent cytotoxicity. A slow degradation of the internalized DNCs by the cells was also observed resulting in the progressive release of the siRNA for up to 6days, as corroborated by laser confocal microscopy. The structural integrity and silencing activity of the double encapsulated siRNA were fully preserved, as demonstrated by HPLC, gel electrophoresis, and potent RNAi activity of siRNA extracted from DNCs. These results demonstrate the potential use of DNCs as a nano drug delivery system for systemic administration and controlled release of siRNA and potentially other sensitive bioactive macromolecules.

Postsynthetic conjugation of RNA to carboxylate and dicarboxylate molecules.

Carboxylates and dicarboxylates are important phosphate mimics. Herein, we present a simple synthetic route for the preparation of RNA carboxylate/dicarboxylate conjugates, starting from suitably protected NH2- and COOH-containing molecules that are coupled to the RNA on the solid support. The key point in our method was the use of trimethylsilylethanol (TMSE-OH) protecting group, which is removed simultaneously with the silyl protecting group on the 2'-OH of the RNA ribose (e.g. t-Butyldimethylsilyl) during the final RNA cleavage/deprotection steps. The usefulness of this method was demonstrated by preparing different RNA-phosphate mimics oligos.

PNA-Rose Bengal Conjugates as Efficient DNA Photomodulators.

Selective photoinduced modulation of DNA may provide a powerful therapeutic tool allowing spatial and temporal control of the photochemical reaction. We have explored the photoreactivity of peptide nucleic acid (PNA) conjugates that were conjugated to a highly potent photosensitizer, Rose Bengal (RB). In addition, a short PEGylated peptide (K-PEG8-K) was conjugated to the C-terminus of the PNA to improve its water solubility. A short irradiation (visible light) of PNA conjugates with a synthetic DNA resulted in highly efficient photomodulation of the DNA as evidenced by polyacrylamide gel electrophoresis (PAGE). In addition, a PNA-RB conjugate replacing K-PEG8-K with four l-glutamic acids (E4) was found to be photoinactive. Irradiation of active PNA-RB conjugates with synthetic DNA in D20 augments the photoactivity; supporting the involvement of singlet oxygen. PAGE, HPLC, and MALDI-TOF analyses indicate that PNA-DNA photo-cross-linking is a significant pathway in the observed photoreactivity. Selective photo-cross-linking of such PNA-RB conjugates may be a novel approach to selective photodynamic therapy (sPDT) as such molecules would be sequence-specific, cell-permeable, and photoactivated in the visible region.

Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

Multi-modal detection of colon malignancy by NIR-tagged recognition polymers and ultrasound contrast agents.

To increase colonoscopy capability to discriminate benign from malignant polyps, we suggest combining two imaging approaches based on targeted polymeric platforms. Water-soluble cationized polyacrylamide (CPAA) was tagged with the near infrared (NIR) dye IR-783-S-Ph-COOH to form Flu-CPAA. The recognition peptide VRPMPLQ (reported to bind specifically to CRC tissues) was then conjugated with the Flu-CPAA to form Flu-CPAA-Pep which was then incorporated into echogenic microbubbles (MBs) made of polylactic acid (PLA) that are highly responsive to ultrasound. The ultimate design includes intravenous administration combined with local ultrasound and intra-colon inspection at the NIR range. In this proof of principle study PLA MBs were prepared by the double emulsion technique and loaded with several types of Flu-CPAA-Pep polymers. After insonation the submicron PLA fragments (SPF)-containing Flu-CPAA-Pep were examined in vitro for their ability to attach to colon cancer cells and in vivo (DMH induced rat model) for their ability to attach to colon malignant tissues and compared to the specific attachment of the free Flu-CPAA-Pep. The generation of SPF-containing Flu-CPAA-Pep resulted in a tissue attachment similar to that of the free, unloaded Flu-CPAA-Pep. The addition of VRPMPLQ to the polymeric backbone of the Flu-CPAA reduced cytotoxicity and improved the specific binding.