Triplex-forming Peptide Nucleic Acid Research Articles

TFOFinder: Python program for identifying purine-only double-stranded stretches in the predicted secondary structure(s) of RNA targets.

Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex-forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex-forming peptide nucleic acid (PNA) oligomers have been used for imaging a structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine) and the design of the corresponding TFO(s). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probe or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication.

Triplex-Forming Peptide Nucleic Acid Controls Dynamic Conformations of RNA Bulges.

RNA folding is driven by the formation of double-helical segments interspaced by loops of unpaired nucleotides. Among the latter, bulges formed by one or several unpaired nucleotides are one of the most common structural motifs that play an important role in stabilizing RNA-RNA, RNA-protein, and RNA-small molecule interactions. Single-nucleotide bulges can fold in alternative structures where the unpaired nucleobase is either looped-out (flexible) in a solvent or stacked-in (intercalated) between the base pairs. In the present study, we discovered that triplex-forming peptide nucleic acids (PNAs) had unusually high affinity for single-purine-nucleotide bulges in double-helical RNA. Depending on the PNA's sequence, the triplex formation shifted the equilibrium between looped-out and stacked-in conformations. The ability to control the dynamic equilibria of RNA's structure will be an important tool for studying structure-function relationships in RNA biology and may have potential in novel therapeutic approaches targeting disease-related RNAs.

Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule

We have developed a new class of triplex-forming peptide nucleic acid (PNA)-based fluorogenic probes for sensing of the panhandle structure of the influenza A virus (IAV) RNA promoter region. Here, a small molecule (DPQ) capable of selectively binding to the internal loop structure was conjugated with triplex-forming forced intercalation of the thiazole orange (tFIT) probe with natural PNA nucleobases. The resulting conjugate, tFIT-DPQ, showed a significant light-up response (83-fold) upon strong (Kd = 107 nM) and structure-selective binding to the IAV RNA promoter region under physiological conditions (pH 7.0, 100 mM NaCl). We demonstrated the conjugation of these two units through the suitable spacer was key to show useful binding and fluorogenic signaling functions. tFIT-DPQ facilitated the sensitive and selective detection of IAV RNA based on its binding to the promoter region. Furthermore, we found that tFIT-DPQ could work as a sensitive indicator for screening of test compounds targeting the IAV RNA promoter region in the fluorescence indicator displacement assay.

Triplex-Forming Peptide Nucleic Acid Probes Having Cyanine Base Surrogates for Fluorogenic Sensing of Double-Stranded RNA

Triplex-Forming Peptide Nucleic Acid Probes Having Cyanine Base Surrogates for Fluorogenic Sensing of Double-Stranded RNA

2-Guanidyl pyridine PNA nucleobase for triple-helical Hoogsteen recognition of cytosine in double-stranded RNA.

In triplex-forming peptide nucleic acid, a novel 2-guanidyl pyridine nucleobase (V) enables recognition of up to two cytosine interruptions in polypurine tracts of dsRNA by engaging the entire Hoogsteen face of C-G base pair. Ab initio and molecular dynamics simulations provided insights into H-bonding interactions that stabilized V·C-G triplets. Our results provided insights for future design of improved nucleobases, which is an important step towards the ultimate goal of recognition of any sequence of dsRNA.

Cellular uptake of 2-aminopyridine-modified peptide nucleic acids conjugated with cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have been extensively used to deliver peptide nucleic acid (PNA) in cells. We have previously found that replacement of cytosine in triplex-forming PNAs with 2-aminopyridine (M) not only enhanced RNA binding, but also improved cellular uptake of PNAs. In this study, we used confocal fluorescence microscopy to evaluate the ability of CPPs to further improve cellular uptake of M-modified PNAs. We found that PNAs conjugated with Tat and octa-arginine peptides were effectively taken up in MCF7 cells when supplied in cell media at 1μM. Remarkably, M-modified PNA without any CPP conjugation also showed strong uptake when the concentration was increased to 5μM. Majority of PNA conjugates remained localized in distinct cytoplasmic vesicles, as judged by dot-like fluorescence patterns. However, M-modified PNAs conjugated with Tat, octa-arginine, and even a simple tri-lysine peptide also showed dispersed fluorescence in cytoplasm and were taken up in nuclei where they localized in larger vesicles, most likely nucleoli. Endosomolytic peptides or chemicals (chloroquine and CaCl2 ) did not release the conjugates from cytosolic vesicles, which suggested that the PNAs were not entrapped in endosomes. We hypothesize that M-modified PNAs escape endosomes and accumulate in cellular compartments rich in RNA, such as nucleoli, stress granules, and P-bodies.

Spectroscopic, thermodynamic and kinetic analysis of selective triplex formation by peptide nucleic acid with double-stranded RNA over its DNA counterpart.

Unlike conventional triplex-forming oligonucleotide (TFO), triplex-forming peptide nucleic acid (PNA) can tightly bind with double-stranded RNA (dsRNA) than double-stranded DNA (dsDNA). Here, we performed spectroscopic, thermodynamic and kinetic experiments for triplex formation by PNA to examine different binding behaviors between PNA - dsRNA and PNA - dsDNA triplexes. We found 9-mer PNA (cytosine content of 66%) formed the thermally stable triplex with dsRNA compared to dsDNA over a wide range of pH (5.5-8.0), salt concentration (50-500 mM NaCl). Both the calorimetric binding constant and the association rate constant for dsRNA were larger than those for dsDNA, indicating the favorable association process for the PNA - dsRNA triplex formation. Comparison with the DNA/RNA heteroduplexes revealed that the DNA strand was detrimental to the triplex stability for PNA, a contrasting result for conventional TFO. The keys underlying the difference in the triplex formation of PNA with different duplexes appear to be the conformational adoptability and the geometric compatibility of PNA to fit the deep, narrow major groove of dsRNA and the helical rigidity difference of the duplexes. Our results emphasize the importance of both the sugar puckering of the duplex and the appropriate conformational flexibility of PNA for the triplex formation.

Triple-Helical Binding of Peptide Nucleic Acid Inhibits Maturation of Endogenous MicroRNA-197.

Sequence specific recognition and functional inhibition of biomedically relevant double-helical RNAs is highly desirable but remains a formidable problem. The present study demonstrates that electroporation of a triplex-forming peptide nucleic acid (PNA), modified with 2-aminopyridine (M) nucleobases, inhibited maturation of endogenous microRNA-197 in SH-SY5Y cells, while having little effect on maturation of microRNA-155 or -27a. In vitro RNA binding and Dicer inhibition assays suggested that the observed biological activity was most likely due to a sequence-specific PNA-RNA triplex formation that inhibited the activity of endonucleases responsible for microRNA maturation. The present study is the first example of modulation of activity of endogenous noncoding RNA using M-modified triplex-forming PNA.

Triplex-Forming Peptide Nucleic Acids with Extended Backbones.

Peptide nucleic acid (PNA) forms a triple helix with double-stranded RNA (dsRNA) stabilized by a hydrogen-bonding zipper formed by PNA's backbone amides (N-H) interacting with RNA phosphate oxygens. This hydrogen-bonding pattern is enabled by the matching ∼5.7 Å spacing (typical for A-form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one -CH2 - group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B-form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA-DNA triplexes compared to PNA-RNA. To test this hypothesis, we synthesized triplex-forming PNAs that had the pseudopeptide backbones extended by an additional -CH2 - group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A-form-like conformations when binding double-stranded nucleic acids. It appears that the original six-atom-long PNA backbone is an almost perfect fit for binding to A-form nucleic acids.

Correction to "Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA".

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionNEXTORIGINAL ARTICLEThis notice is a correctionCorrection to “Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA”Takaya Sato, Yusuke Sato, and Seiichi Nishizawa*Cite this: J. Am. Chem. Soc. 2017, 139, 9, 3567Publication Date (Web):February 27, 2017Publication History Published online27 February 2017Published inissue 8 March 2017https://doi.org/10.1021/jacs.7b01636Copyright © 2017 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1443Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (125 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Optimization of the Alkyl Linker of TO Base Surrogate in Triplex-Forming PNA for Enhanced Binding to Double-Stranded RNA.

A series of triplex-forming peptide nucleic acid (TFP) probes carrying a thiazole orange (TO) base surrogate through an alkyl linker was synthesized, and the interactions between these so-called tFIT probes and purine-rich sequences within double-stranded RNA (dsRNA) were examined. We found that the TO base surrogate linker significantly affected both the binding affinity and the fluorescence response upon triplex formation with the target dsRNA. Among the probes examined, the TO base surrogate connected through the propyl linker in the tFIT probes increased the binding affinity by a factor of ten while maintaining its function as the fluorescent universal base. Isothermal titration calorimetry experiments revealed that the increased binding affinity resulted from the gain in the binding enthalpy, which could be explained by the enhanced π-stacking interaction between the TO base surrogate and the dsRNA part of the triplex. We expect that these results will provide a molecular basis for designing strong binding tFIT probes for fluorescence sensing of various kinds of purine-rich dsRNAs sequences including those carrying a pyrimidine-purine inversion. The obtained data also offers a new insight into further development of the universal bases incorporated in TFP.

Red-emissive triplex-forming PNA probes carrying cyanine base surrogates for fluorescence sensing of double-stranded RNA.

Red-emissive fluorescent probes have been developed by integration of quinoline blue or thiazole red as the base surrogate into triplex-forming PNAs, allowing selective sensing of a sequence of double-stranded RNA.

Incorporating a guanidine-modified cytosine base into triplex-forming PNAs for the recognition of a C-G pyrimidine-purine inversion site of an RNA duplex.

RNA duplex regions are often involved in tertiary interactions and protein binding and thus there is great potential in developing ligands that sequence-specifically bind to RNA duplexes. We have developed a convenient synthesis method for a modified peptide nucleic acid (PNA) monomer with a guanidine-modified 5-methyl cytosine base. We demonstrated by gel electrophoresis, fluorescence and thermal melting experiments that short PNAs incorporating the modified residue show high binding affinity and sequence specificity in the recognition of an RNA duplex containing an internal inverted Watson-Crick C-G base pair. Remarkably, the relatively short PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. The attached guanidine group stabilizes the base triple through hydrogen bonding with the G base in a C-G pair. Selective binding towards an RNA duplex over a single-stranded RNA can be rationalized by the fact that alkylation of the amine of a 5-methyl C base blocks the Watson–Crick edge. PNAs incorporating multiple guanidine-modified cytosine residues are able to enter HeLa cells without any transfection agent.

Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA.

We have developed a new fluorescent sensing probe for double-stranded RNA (dsRNA) by integrating thiazole orange (TO) as a base surrogate into triplex-forming PNA. Our probe forms the thermally stable triplex with the target dsRNA at acidic pH; and the triplex formation is accompanied by the remarkable light-up response of the TO unit. The binding of our probe to the target dsRNA proceeds very rapidly, allowing real-time monitoring of the triplex formation. Importantly, we found the TO base surrogate in our probe functions as a universal base for the base pair opposite the TO unit in the triplex formation. Furthermore, the TO unit is significantly more responsive for the fully matched dsRNA sequence compared to the mismatch-containing sequences, which enables the analysis of the target dsRNA sequence at the single-base pair resolution. The binding and sensing functions of our probe are described for the development of fluorescent probes applicable to sensing biologically relevant dsRNA.

Triplex-forming peptide nucleic acid modified with 2-aminopyridine as a new tool for detection of A-to-I editing

RNA editing from adenosine to inosine (A-to-I editing) is one of the mechanisms that regulate and diversify the transcriptome. Here, a triplex-forming peptide nucleic acid (PNA) modified with a 2-aminopyridine nucleobase was applied for the recognition of the A-to-I editing event in double-stranded RNAs. The triplex-forming PNA enabled sequence-specific detection of single nucleobase editing at sub-nanomolar concentration.

Correction: Triplex-forming peptide nucleic acid modified with 2-aminopyridine as a new tool for detection of A-to-I editing.

Correction for 'Triplex-forming peptide nucleic acid modified with 2-aminopyridine as a new tool for detection of A-to-I editing' by Chiara Annoni et al., Chem. Commun., 2016, 52, 7935-7938.

Triplex-forming PNA modified with unnatural nucleobases: the role of protonation entropy in RNA binding

Peptide nucleic acid (PNA) modified with unnatural nucleobases enables the formation of a highly stable triplex with a double-stranded RNA at physiological pH. In this communication, we evaluated kinetics and thermodynamics of PNA/RNA triplex formation as a function of both pH and temperature. Protonation entropy was found to be the major factor responsible for the destabilization of the triplex and for the progressive decrease in the association rate at more basic pHs.

Nucleobase-Modified PNA Suppresses Translation by Forming a Triple Helix with a Hairpin Structure in mRNA In Vitro and in Cells.

Compounds that bind specifically to double-stranded regions of RNA have potential as regulators of structure-based RNA function; however, sequence-selective recognition of double-stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA-RNA triplexes. Herein, we demonstrate that a 9-mer PNA forms a sequence-specific PNA-RNA triplex with a dissociation constant of less than 1 nm at physiological pH. The triplex formed within the 5' untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex-forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double-stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein-RNA interactions.

Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here, we use triplex-forming PNA molecules and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep sequencing reveals negligible off-target effects in partially homologous sites. Intranasal application of nanoparticles in CF mice produces changes in nasal epithelium potential differences consistent with corrected CFTR, with gene correction also detected in lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.