Biophysical evaluation of antiparallel triplexes for biosensing and biomedical applications.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

In Vitro Approaches to Explore the Anticancer Potential of One Natural Flavanone and Four Derivatives Loaded in Biopolymeric Nanoparticles for Application in Topical Delivery Treatments.

The increasing number of skin cancer cases worldwide and the adverse side effects of current treatments have led to the search for new anticancer agents. In this present work, the anticancer potential of the natural flavanone 1, extracted from Eysenhardtia platycarpa, and four flavanone derivatives 1a-d obtained by different reactions from 1 was investigated by an in silico study and through cytotoxicity assays in melanoma (M21), cervical cancer (HeLa) cell lines and in a non-tumor cell line (HEK-293). The free compounds and compounds loaded in biopolymeric nanoparticles (PLGA NPs 1, 1a-d) were assayed. A structure-activity study (SAR) was performed to establish the main physicochemical characteristics that most contribute to cytotoxicity. Finally, ex vivo permeation studies were performed to assess the suitability of the flavanones for topical administration. Results revealed that most of the studied flavanones and their respective PLGA NPs inhibited cell growth depending on the concentration; 1b should be highlighted. The descriptors of the energetic factor were those that played a more important role in cellular activity. PLGA NPs demonstrated their ability to penetrate (Qp of 17.84-118.29 µg) and be retained (Qr of 0.01-1.44 g/gskin/cm2) in the skin and to exert their action for longer. The results of the study suggest that flavanones could offer many opportunities as a future anticancer topical adjuvant treatment.

In Vitro and In Vivo Effects of the Combination of Polypurine Reverse Hoogsteen Hairpins against HER-2 and Trastuzumab in Breast Cancer Cells.

Therapeutic oligonucleotides are powerful tools for the inhibition of potential targets involved in cancer. We describe the effect of two Polypurine Reverse Hoogsteen (PPRH) hairpins directed against the ERBB2 gene, which is overexpressed in positive HER-2 breast tumors. The inhibition of their target was analyzed by cell viability and at the mRNA and protein levels. The combination of these specific PPRHs with trastuzumab was also explored in breast cancer cell lines, both in vitro and in vivo. PPRHs designed against two intronic sequences of the ERBB2 gene decreased the viability of SKBR-3 and MDA-MB-453 breast cancer cells. The decrease in cell viability was associated with a reduction in ERBB2 mRNA and protein levels. In combination with trastuzumab, PPRHs showed a synergic effect in vitro and reduced tumor growth in vivo. These results represent the preclinical proof of concept of PPRHs as a therapeutic tool for breast cancer.

Trioleyl Pyridinium, a Cationic Transfection Agent for the Lipofection of Therapeutic Oligonucleotides into Mammalian Cells.

BACKGROUND: One of the most significant limitations that therapeutic oligonucleotides present is the development of specific and efficient delivery vectors for the internalization of nucleic acids into cells. Therefore, there is a need for the development of new transfection agents that ensure a proper and efficient delivery into mammalian cells. METHODS: We describe the synthesis of 1,3,5-tris[(4-oelyl-1-pyridinio)methyl]benzene tribromide (TROPY) and proceeded to the validation of its binding capacity toward oligonucleotides, the internalization of DNA into the cells, the effect on cell viability, apoptosis, and its capability to transfect plasmid DNA. RESULTS: The synthesis and chemical characterization of TROPY, which can bind DNA and transfect oligonucleotides into mammalian cells through clathrin and caveolin-mediated endocytosis, are described. Using a PPRH against the antiapoptotic survivin gene as a model, we validated that the complex TROPY-PPRH decreased cell viability in human cancer cells, increased apoptosis, and reduced survivin mRNA and protein levels. TROPY was also able to stably transfect plasmid DNA, as demonstrated by the formation of viable colonies upon the transfection of a dhfr minigene into dhfr-negative cells and the subsequent metabolic selection. CONCLUSIONS: TROPY is an efficient transfecting agent that allows the delivery of therapeutic oligonucleotides, such as PPRHs and plasmid DNA, inside mammalian cells.

On the uptake of cationic liposomes by cells: From changes in elasticity to internalization.

In this study, we assessed the capacity of a previously reported engineered liposomal formulation, which had been tested against model membranes mimicking the lipid composition of the HeLa plasma membrane, to fuse and function as a nanocarrier in cells. We used atomic force microscopy to observe physicochemical changes on the cell surface and confocal microscopy to determine how the liposomes interact with cell membranes and released their load. In addition, we performed viability assays using methotrexate as an active drug to obtain proof of concept of the formulation´s capacity to function as a drug delivery-system. The interaction of engineered liposomes with living cells corroborates the information obtained using model membranes and supports the capacity of the engineered liposomal formulation to serve as a potential nanocarrier.

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA).

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

Base-Mediated Nitrophenyl Reductive Cyclization for the Synthesis of Hexahydro-2,6-methano-1-benzazocines.

A Diels-Alder reaction leading to 4-nitrophenylcyclohexanones followed by a newly developed base-mediated reductive cyclization of the resulting ketone tethered to the nitrobenzene moiety gives access to the hexahydro-2,6-methano-1-benzazocine ring system present in several biologically interesting natural products such as aspernomine. The scope of the reaction was explored with eight substituted nitrobenzenes, obtaining yields of up to 87%. The highest cytotoxicity was observed in benzazocine 4h, bearing an enone moiety, which was active against eight cancer cell lines.

Targeting KRAS Regulation with PolyPurine Reverse Hoogsteen Oligonucleotides.

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.

Targeting MYC Regulation with Polypurine Reverse Hoogsteen Oligonucleotides.

The oncogene MYC has key roles in transcription, proliferation, deregulating cellular energetics, and more. Modulating the expression or function of the MYC protein is a viable therapeutic goal in an array of cancer types, and potential inhibitors of MYC with high specificity and selectivity are of great interest. In cancer cells addicted to their aberrant MYC function, suppression can lead to apoptosis, with minimal effects on non-addicted, non-oncogenic cells, providing a wide therapeutic window for specific and efficacious anti-tumor treatment. Within the promoter of MYC lies a GC-rich, G-quadruplex (G4)-forming region, wherein G4 formation is capable of mediating transcriptional downregulation of MYC. Such GC-rich regions of DNA are prime targets for regulation with Polypurine Reverse Hoogsteen hairpins (PPRHs). The current study designed and examined PPRHs targeting the G4-forming and four other GC-rich regions of DNA within the promoter or intronic regions. Six total PPRHs were designed, examined in cell-free conditions for target engagement and in cells for transcriptional modulation, and correlating cytotoxic activity in pancreatic, prostate, neuroblastoma, colorectal, ovarian, and breast cancer cells. Two lead PPRHs, one targeting the promoter G4 and one targeting Intron 1, were identified with high potential for further development as an innovative approach to both G4 stabilization and MYC modulation.

PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing.

PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs.

Synthesis and validation of DOPY: A new gemini dioleylbispyridinium based amphiphile for nucleic acid transfection.

Nucleic acids therapeutics provide a selective and promising alternative to traditional treatments for multiple genetic diseases. A major obstacle is the development of safe and efficient delivery systems. Here, we report the synthesis of the new cationic gemini amphiphile 1,3-bis[(4-oleyl-1-pyridinio)methyl]benzene dibromide (DOPY). Its transfection efficiency was evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs), a nucleic acid tool for gene silencing and gene repair developed in our laboratory. The interaction of DOPY with PPRHs was confirmed by gel retardation assays, and it forms complexes of 155 nm. We also demonstrated the prominent internalization of PPRHs using DOPY compared to other chemical vehicles in SH-SY5Y, PC-3 and DF42 cells. Regarding gene silencing, a specific PPRH against the survivin gene delivered with DOPY decreased survivin protein levels and cell viability more effectively than with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) in both SH-SY5Y and PC-3 cells. We also validated the applicability of DOPY in gene repair approaches by correcting a point mutation in the endogenous locus of the dhfr gene in DF42 cells using repair-PPRHs. All these results indicate both an efficient entry and release of PPRHs at the intracellular level. Therefore, DOPY can be considered as a new lipid-based vehicle for the delivery of therapeutic oligonucleotides.

Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells.

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. METHODS: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. RESULTS: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. CONCLUSIONS: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

Nucleic acids therapeutics using PolyPurine Reverse Hoogsteen hairpins.

PolyPurine Reverse Hoogsteen hairpins (PPRHs) are DNA hairpins formed by intramolecular reverse Hoogsteen bonds which can bind to polypyrimidine stretches in dsDNA by Watson:Crick bonds, thus forming a triplex and displacing the fourth strand of the DNA complex. PPRHs were first described as a gene silencing tool in vitro for DHFR, telomerase and survivin genes. Then, the effect of PPRHs directed against the survivin gene was also determined in vivo using a xenograft model of prostate cancer cells (PC3). Since then, the ability of PPRHs to inhibit gene expression has been explored in other genes involved in cancer (BCL-2, mTOR, topoisomerase, C-MYC and MDM2), in immunotherapy (SIRPα/CD47 and PD-1/PD-L1 tandem) or in replication stress (WEE1 and CHK1). Furthermore, PPRHs have the ability to target the complementary strand of a G-quadruplex motif as a regulatory element of the TYMS gene. PPRHs have also the potential to correct point mutations in the DNA as shown in two collections of CHO cell lines bearing mutations in either the dhfr or aprt loci. Finally, based on the capability of PPRHs to form triplexes, they have been incorporated as probes in biosensors for the determination of the DNA methylation status of PAX-5 in cancer and the detection of mtLSU rRNA for the diagnosis of Pneumocystis jirovecii. Of note, PPRHs have high stability and do not present immunogenicity, hepatotoxicity or nephrotoxicity in vitro. Overall, PPRHs constitute a new economical biotechnological tool with multiple biomedical applications.

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins.

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.

Targeting replication stress response using polypurine reverse hoogsteen hairpins directed against WEE1 and CHK1 genes in human cancer cells.

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.

Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells.

In this study, we describe the correction of single-point mutations in mammalian cells by repair-polypurine reverse Hoogsteen hairpins (repair-PPRHs). These molecules consist of (1) a PPRH hairpin core that binds to a polypyrimidine target sequence in the double-stranded DNA (dsDNA), producing a triplex structure, and (2) an extension sequence homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutation and acting as a donor DNA to correct the mutation. We repaired different point mutations in the adenosyl phosphoribosyl transferase (aprt) gene contained in different aprt-deficient Chinese hamster ovary (CHO) cell lines. Because we had previously corrected mutations in the dihydrofolate reductase (dhfr) gene, in this study, we demonstrate the generality of action of the repair-PPRHs. Repaired cells were analyzed by DNA sequencing, mRNA expression, and enzymatic activity to confirm the correction of the mutation. Moreover, whole-genome sequencing analyses did not detect any off-target effect in the repaired genome. We also performed gel-shift assays to show the binding of the repair-PPRH to the target sequence and the formation of a displacement-loop (D-loop) structure that can trigger a homologous recombination event. Overall, we demonstrate that repair-PPRHs achieve the permanent correction of point mutations in the dsDNA at the endogenous level in mammalian cells without off-target activity.

Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology.

Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.

A novel DNA-binding motif in prostate tumor overexpressed-1 (PTOV1) required for the expression of ALDH1A1 and CCNG2 in cancer cells.

PTOV1 is a transcription and translation regulator and a promoter of cancer progression. Its overexpression in prostate cancer induces transcription of drug resistance and self-renewal genes, and docetaxel resistance. Here we studied PTOV1 ability to directly activate the transcription of ALDH1A1 and CCNG2 by binding to specific promoter sequences. Chromatin immunoprecipitation and electrophoretic mobility shift assays identified a DNA-binding motif inside the PTOV-A domain with similarities to known AT-hooks that specifically interacts with ALDH1A1 and CCNG2 promoters. Mutation of this AT-hook-like sequence significantly decreased the expression of ALDH1A1 and CCNG2 promoted by PTOV1. Immunohistochemistry revealed the association of PTOV1 with mitotic chromosomes in high grade prostate, colon, bladder, and breast carcinomas. Overexpression of PTOV1, ALDH1A1, and CCNG2 significantly correlated with poor prognosis in prostate carcinomas and with shorter relapse-free survival in colon carcinoma. The previously described interaction with translation complexes and its direct binding to ALDH1A1 and CCNG2 promoters found here reveal the PTOV1 capacity to modulate the expression of critical genes at multiple levels in aggressive cancers. Remarkably, the AT-hook motifs in PTOV1 open possibilities for selective targeting its nuclear and/or cytoplasmic activities.

Cancer immunotherapy using PolyPurine Reverse Hoogsteen hairpins targeting the PD-1/PD-L1 pathway in human tumor cells.

Immunotherapy approaches stand out as innovative strategies to eradicate tumor cells. Among them, PD-1/PD-L1 immunotherapy is considered one of the most successful advances in the history of cancer immunotherapy. We used our technology of Polypurine reverse Hoogsteen hairpins (PPRHs) for silencing both genes with the aim to provoke the elimination of tumor cells by macrophages in co-culture experiments. Incubation of PPRHs against PD-1 and PD-L1 decreased the levels of mRNA and protein in THP-1 monocytes and PC3 prostate cancer cells, respectively. Viability of THP-1 cells and macrophages obtained by PMA-differentiation of THP-1 cells was not affected upon incubation with the different PPRHs. On the other hand, PC3 cell survival was partially decreased by PPRHs against PD-L1. The greatest effect in decreasing cell viability was obtained in macrophages/PC3 co-culture experiments by combining PPRHs against PD-1 and PD-L1. This effect was also observed in other cancer cell lines: HeLa, SKBR3 and to a minor extent in M21. Apoptosis was not detected when macrophages were treated with the different PPRHs. However, co-cultures of macrophages with the four cancer cell lines treated with PPRHs showed an increase in apoptosis. The order of fold-increase in apoptosis was HeLa > PC3 > SKBR3 > M21. This study demonstrates that PPRHs could be powerful pharmacological agents to use in immunotherapy approaches for the inhibition of PD-1 and PD-L1.