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Targeting the genes regulate the lineage commitment of human mesenchymal stem cells (hMSCs) to adipocytes provides a promising strategy for addressing obesity. In this study, we investigated the therapeutic potential of cell-penetrating anti-sense peptide nucleic acids (PNAs) designed to enhance solubility and hybridization properties, specifically targeting sulfatase 2 (SULF2), a potential reciprocal regulator of adipocyte and osteoblast differentiation in hMSCs. Cell-penetrating modified PNA oligomers effectively inhibit SULF2 gene transcription, leading to significant reductions in adiponectin protein synthesis and intracellular lipid droplet accumulation during adipogenesis in human bone marrow-derived MSCs (hBM-MSCs). Notably, PNA oligomer compound 5 exhibited the most potent anti-adipogenic activity, with an IC 50 value of 0.28 μM. These findings show the potential of SULF2-targeting cell-penetrating PNA oligomers as novel therapeutic agents for obesity-related metabolic diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daram Jung reports financial support was provided by OliPass Corporation. Yeasel Jeon reports financial support was provided by OliPass Corporation. Minhee Kim reports financial support was provided by Minhee Kim. Areum Kim reports was provided by OliPass Corporation. Minsoo Noh, Ph.D. reports a relationship with Soul National University that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We have explored the reaction of a three-components mixture of aminomalononitrile, urea and α-amino acid methyl esters for the multicomponent synthesis substituted purines resembling PNA's building blocks. 2,6-diamino-purines, 6-amino-3,9-dihydro-2H-purin-2-one (iso-guanines), and 3,9-dihydro-6H-purin-6-one derivatives, selectively decorated at C(8)-position of the purine ring with different amino acid residues, were obtained from acceptable to good yields. The regio-selectivity of the transformation was controlled by the use of urea in the ternary mixture and by the annulation agent involved in the ring-closure of amino-imidazole carbonitrile intermediates. Solvent free conditions, microwave irradiation and simple one-carbon containing reagents further satisfied the major requirement of atom economy and sustainable chemistry. Due to the prebiotic nature of the three-components mixture and of annulation agents, it also embodies the possibility for the synthesis of novel PNAs bearing purine nucleobases decorated at C(8)-position of the imidazole ring as alternative RNA analogues in molecular evolution., (© 2024 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

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 (λ em, 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-CH 2 COOH 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.

Advantages like biocompatibility, biodegradability and tunability allowed the exploitation of peptides and peptidomimetics as versatile therapeutic or diagnostic agents. Because of their selectivity towards transmembrane receptors or cell membranes, peptides have also been identified as suitable molecules able to deliver in vivo macromolecules, proteins or nucleic acids. However, after the identification of the homodimer diphenylalanine (FF) as an aggregative motif inside the Aβ 1-42 polypeptide, short and ultrashort peptides have been studied as building blocks for the fabrication of supramolecular, ordered nanostructures for applications in biotechnological, biomedical and industrial fields. In this perspective, many hybrid molecules that combine FF with other chemical entities have been synthesized and characterized. Two novel hybrid derivatives (tFaF and cFgF), in which the FF homodimer is alternated with the peptide-nucleic acid (PNA) heterodimer "g-c" (guanine-cytosine) or "a-t" (adenine-thymine) and their dimeric forms (tFaF) 2 and (cFgF) 2 were synthesized. The structural characterization performed by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopies highlighted the capability of all the FF-PNA derivatives to self-assemble into β-sheet structures. As a consequence of this supramolecular organization, the resulting aggregates also exhibit optoelectronic properties already reported for other similar nanostructures. This photoemissive behavior is promising for their potential applications in bioimaging., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

2,4-Difluorotoluene is a nonpolar isostere of thymidine that has been used as a powerful mechanistic probe to study the role of hydrogen bonding in nucleic acid recognition and interactions with polymerases. In the present study, we evaluated five fluorinated benzenes as nucleobase analogues in peptide nucleic acids designed for triple helical recognition of double helical RNA. We found that analogues having para and ortho fluorine substitution patterns (as in 2,4-difluorotoluene) selectively stabilized Hoogsteen triplets with U-A base pairs. The results were consistent with attractive electrostatic interactions akin to non-canonical F to H-N and C-H to N hydrogen bonding. The fluorinated nucleobases were not able to stabilize Hoogsteen-like triplets with pyrimidines in either G-C or A-U base pairs. Our results illustrate the ability of fluorine to engage in non-canonical base pairing and provide insights into triple helical recognition of RNA., (© 2021 Wiley-VCH GmbH.)

Sequence specific recognition of regulatory noncoding RNAs would open new possibilities for fundamental science and medicine. However, molecular recognition of such complex double-stranded RNA (dsRNA) structures remains a formidable problem. Recently, we discovered that peptide nucleic acids (PNAs) form an unusually stable and sequence-specific triple helix with dsRNA. Triplex-forming PNAs could become universal tools for recognition of noncoding dsRNAs but are limited by the requirement of polypurine tracts in target RNAs as only purines form stable Hoogsteen hydrogen bonded base triplets. Herein, we systematically surveyed simple nitrogen heterocycles P N as modified nucleobases for recognition of cytosine in P N *C-G triplets. We found that a 3-pyridazinyl nucleobase formed significantly more stable P N *C-G triplets than other heterocycles including the pyrimidin-2-one previously used by us and others for recognition of cytosine interruptions in polypurine tracts of PNA-dsRNA triplexes. Our results improve triple helical recognition of dsRNA and provide insights for future development of new nucleobases to expand the sequence scope of noncoding dsRNAs that can be targeted by triplex-forming PNAs.

We report the synthesis of a peptide nucleic acid (PNA) monomer containing preQ 1 , a positively charged guanine analogue. The new monomer was incorporated into PNA oligomers using standard Fmoc-chemistry-based solid-phase synthesis. The preQ 1 unit-containing PNA oligomers exhibited improved affinity for their complementary DNA through electrostatic attraction, and their sequence specificity was not compromised. It could be beneficial to incorporate preQ 1 into PNA oligomers instead of guanine when creating antisense/antigene agents or research tools., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A novel synthesis of C(2)-modified peptide nucleic acids (PNAs) is proposed, using a submonomeric strategy with minimally protected building blocks, which allowed a reduction in the required synthetic steps. N(3)-unprotected, d-Lys- and d-Arg-based backbones were used to obtain positively charged PNAs with high optical purity, as inferred from chiral GC measurements. "Chiral-box" PNAs targeting the G12D point mutation of the KRAS gene were produced using this method, showing improved sequence selectivity for the mutated- vs wild-type DNA strand with respect to unmodified PNAs.

DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labelling by way of toehold-mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.

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 -CH 2 - 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 -CH 2 - 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., (© 2020 Wiley-VCH GmbH.)

Bimodal PNAs are new PNA constructs designed to bind two different cDNA sequences synchronously to form double duplexes. They are synthesized on solid phase using sequential coupling and click reaction to introduce a second base in each monomer at C α via alkyltriazole linker. The ternary bimodal PNA:DNA complexes show stability higher than that of individual duplexes. Bimodal PNAs are appropriate to create higher-order fused nucleic acid assemblies.

We propose a novel, ultrasensitive and low-cost sensor using reversible addition-fragmentation chain transfer (RAFT) polymerization as a signal amplification strategy for the detection of CYFRA 21-1 DNA fragment, a tumor marker of non-small cell lung carcinoma. The peptide nucleic acid (PNA) probes were firstly immobilized on magnetic beads (MBs) to capture the CYFRA 21-1 DNA specifically. After hybridization, CPAD was tethered to the hetero duplexes through carboxylate-Zr 4+ -phosphate chemistry. Subsequently, a number of fluorescent tags were introduced to the heteroduplexes through RAFT polymerization, leading to an amplification of the fluorescence signal. The sensor demonstrates a low limit of detection (LOD) of 0.02 fM. It has great selectivity with respect to base mismatch DNA, and high anti-interference ability in normal human serum. Overall findings of the study suggest that proposed sensor holds enormous potential to be used as a tool for the early-stage diagnosis of lung cancers.

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Pyrrolidinyl PNA with an α-/β-dipeptide backbone consisting of alternating nucleobase-modified D-proline and (1S,2S)-2-aminocyclopentanecarboxylic acid (also known as acpcPNA) is a class of conformationally constrained PNA that shows exceptional DNA hybridization properties including very high specificity and the inability to form self-pairing hybrids. In this chapter, details of the syntheses of acpcPNA as well as its monomers and a protocol for site-specific labeling with a fluorescent dye via click chemistry are reported.

Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described.

Exploration of PNA-peptide conjugates as potential antisense antibiotics necessitates a fast and efficient synthesis protocols for amounts that facilitate determination of structure-activity relationships and in vivo studies in animal infection models. Fmoc/Boc-protected PNA monomers are here used for assembly of oligomers by optimized protocols involving either a manual synthesis method at room temperature or automated microwave-assisted coupling of monomers on a peptide synthesizer.

Because of the important roles noncoding RNAs play in gene expression, their sequence-specific recognition is important for both fundamental science and the pharmaceutical industry. However, most noncoding RNAs fold in complex helical structures that are challenging problems for molecular recognition. Herein, we describe a method for sequence-specific recognition of double-stranded RNA using peptide nucleic acids (PNAs) that form triple helices in the major grove of RNA under physiologically relevant conditions. We also outline methods for solid-phase conjugation of PNA with cell-penetrating peptides and fluorescent dyes. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

Peptide nucleic acids (PNAs) can be modified with aliphatic lipid chains and designed to be water soluble and able to spontaneously insert into phospholipid bilayers. Liposomes with 1.5% negatively charged POPG can be driven to fuse and mix their inner content volumes via functionalization with such lipidated peptide nucleic acids (LiPNAs). During fusion, only low amounts of leakage occur (<5%). We describe here the synthesis and purification of such LiPNAs using an automated peptide synthesizer and the preparation of LiPNA functionalized liposomes. Further, we describe the measurement of LiPNA-induced fusion using a fluorescence-based assay for the content mixing between a liposome population with an encapsulated self-quenching fluorescent dye (SRB) and a buffer-filled liposome population.

Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins.

Synthesis of exclusive N 2 -(isobutyryl)-9-(carboxymethyl)guanine, an important moiety for peptide nucleic acid synthesis has been reported through a high-yielding reaction scheme starting from 6-chloro-2-amino purine. Crystal structures of two intermediates confirmed the formation of N 9-regioisomer. This new synthetic route can potentially replace the conventional tedious method with moderate overall yield.

Using near-infrared fluorophore Alexa Fluor 680 labeled peptide nucleic acids (PNAs) the biodistribution of such antisense agents can be analyzed in real time in live mice using in vivo imaging. Using the fluorescence intensity emitted from the mouse at different time points following administration, the systemic distribution and organ accumulation of PNA can be tracked. In addition, an estimation of the body half-life of the compound can be obtained by the change in fluorescence intensity over time. With this technique, the distribution of compounds can be monitored real time, while reducing the number of animals and amount of compounds required.

We report the syntheses of chemical building blocks of a particular class of chiral PNAs, called miniPEG-containing (R)-gamma PNAs (or (R)-MPγPNAs). The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of racemization in the alkylation and reductive amination steps. The methodology is general and robust, ideally suited for large-scale monomer productions with most synthetic steps providing excellent chemical yields without the need for purification other than a simple workup and precipitation.

Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.

DNA-encoded library technologies have emerged as a powerful platform to rapidly screen for binders to a protein of interest. These technologies are underpinned by the ability to encode a rich diversity of small molecules. While large libraries are accessible by cycles of mix and split synthesis, libraries based on single chemistries tend to be redundant. Furthermore, the quality of libraries generally decreases with the number of synthetic transformations performed in its synthesis. An alternative approach is to use hybridization to program the combinatorial assembly of fragment pairs onto a library of DNA templates. A broad molecular diversity is more easily sampled since it arises from the pairing of diverse fragments. Upon identification of productive fragment pairs, a focused library covalently linking the fragments is prepared. This focused library includes linker of different length and geometry and offers the opportunity to enrich the selected fragment set with close neighbors. Herein we describe detailed protocols to covalently link diverse fragments and screen fragment-based libraries using commercially available microarray platform.

Nucleic acids and proteins are the fundamental biopolymers that support all life on Earth. Nucleic acids store large amounts of information in nucleobase sequences while peptides and proteins utilize diverse amino acid functional groups to adopt complex structures and perform wide-ranging activities. Although nature has evolved machinery to read the nucleic acid code and translate it into amino acid code, the extant biopolymers are restricted to encoding amino acid or nucleotide sequences separately, limiting their potential applications in medicine and biotechnology. Here we describe the design, synthesis, and stimuli-responsive assembly behavior of a bilingual biopolymer that integrates both amino acid and nucleobase sequences into a single peptide nucleic acid (PNA) scaffold to enable tunable storage and retrieval of tertiary structural behavior and programmable molecular recognition capabilities. Incorporation of a defined sequence of amino acid side-chains along the PNA backbone yields amphiphiles having a "protein code" that directs self-assembly into micellar architectures in aqueous conditions. However, these amphiphiles also carry a "nucleotide code" such that subsequent introduction of a complementary RNA strand induces a sequence-specific disruption of assemblies through hybridization. Together, these properties establish bilingual PNA as a powerful biopolymer that combines two information systems to harness structural responsiveness and sequence recognition. The PNA scaffold and our synthetic system are highly generalizable, enabling fabrication of a wide array of user-defined peptide and nucleotide sequence combinations for diverse future biomedical and nanotechnology applications.

Triple-helix formation, using Hoogsteen hydrogen bonding of triplex-forming oligonucleotides, represents an attractive method for sequence-specific recognition of double-stranded nucleic acids. However, practical applications using triple-helix-forming oligonucleotides and their analogues are limited to long homopurine sequences. The key problem for recognition of pyrimidines is that they present only one hydrogen-bond acceptor or donor group in the major groove. Herein, we report our first attempt to overcome this problem by using peptide nucleic acids (PNAs) modified with extended nucleobases that form three hydrogen bonds along the entire Hoogsteen edge of the Watson-Crick base pair. New nucleobase triples (five) were designed, and their hydrogen bonding feasibility was confirmed by ab initio calculations. PNA monomers carrying the modified nucleobases were synthesized and incorporated in short model PNA sequences. Isothermal titration calorimetry showed that these nucleobases had a modest binding affinity for their double-stranded RNA (dsRNA) targets. Finally, molecular modeling of the modified triples in PNA-dsRNA helix suggested that the modest binding affinity was caused by subtle structural deviations from ideal hydrogen-bonding arrangements or disrupted π-stacking of the extended nucleobase scaffolds.

MicroRNAs are a ubiquitous class of non-coding RNAs able to regulate gene expression in diverse biological processes. Widespread miRNAs deregulation was reported in numerous diseases including cancer, with several miRNAs playing oncogenic and/or tumor suppressive role by targeting multiple mRNAs simultaneously. Based on these findings, miRNAs have emerged as promising therapeutic tools for cancer treatment. Herein, for the first time, peptide nucleic acids (PNAs) were studied to develop a new class of molecules able to target 3'UTR on MYCN mRNA without a fully complementary base pairing sequence (as miRNAs). For our proof of concept study we have selected as a model the miRNA-34a, which acts as a tumor suppressor in a number of cancers including neuroblastoma. In particular, miRNA-34a is a direct regulator of MYCN oncogene, whose overexpression is a prominent biomarker for the highly aggressive neuroblastoma phenotype. The design and synthesis of three PNA-based oligomers of different length was described, and their interaction with two binding sites on the target MYCN mRNA was investigated by molecular dynamics simulation, and spectroscopic techniques (CD, UV). Intake assay and confocal microscopy of PNA sequences were also carried out in vitro on neuroblastoma Kelly cells. Despite the presence of multiple mismatches, the PNA/RNA hetero duplexes retain very interesting features in terms of stability, affinity as well as of cellular uptake., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Peptide nucleic acids array technology is a method of greatly increasing the throughput of laboratory processes to efficiently perform large-scale genetic tests. Diethylene glycol-containing chiral γPNA (miniPEG-γPNA) is considered to be the best PNA derivative and one of the best candidates for gene detection, because it can hybridize DNA with greater affinity and sequence selectivity than DNA and ordinary aminoethylglycyl PNA (aegPNA). Herein, miniPEG-γPNA probes are synthesized by 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS) in a mild condition, and a new biochip fabrication method "Film-Spotting" is invented, by which γPNA arrays with regular pattern, uniform luminance, and very low fluorescence background are obtained easily and cheaply. The miniPEG-γPNA array can effectively distinguish the full matched and mismatched targets in SSarc buffer, serum and urine, and the detection limit of complementary DNA is less than 5.97 nM. A miniPEG-γPNA array for BRCA1 gene mutation (3099delT) detection is also fabricated with a very good detection performance. This work provides an effective avenue for the diagnosis of breast cancer biomarker and expands the application of miniPEG-γPNA in the field of biochip., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We report herein the synthesis and DNA/RNA binding properties of bPNA+, a new variant of bifacial peptide nucleic acid (bPNA) that binds oligo T/U nucleic acids to form triplex hybrids. By virtue of a new bivalent side chain on bPNA+, similar DNA affinity and hybrid thermostability can be obtained with half the molecular footprint of previously reported bPNA. Lysine derivatives bearing two melamine bases (K 2M ) can be prepared on multigram scale by double reductive alkylation with melamine acetaldehyde, resulting in a tertiary amine side chain that affords both peptide solubility and selective base-triple formation with 4 T/U bases; the Fmoc-K 2M derivative can be used directly in solid phase peptide synthesis, rendering bPNA+ conveniently accessible. A compact bPNA+binding site of two U6 domains can be genetically encoded to replace existing 6 bp stem elements at virtually any location within an RNA transcript. We thus replaced internal 6 bp RNA stems that supported loop regions with 6 base-triple hybrid stems using fluorophore-labeled bPNA+. As the loop regions engaged in RNA tertiary interactions, the labeled hybrid stems provided a fluorescent readout; bPNA+ enabled this readout without covalent chemical modification or introduction of new structural elements. This strategy was demonstrated to be effective for reporting on widely observed RNA tertiary interactions such as intermolecular RNA-RNA kissing loop dimerization, RNA-protein binding, and intramolecular RNA tetraloop-tetraloop receptor binding, illustrating the potential general utility of this method. The modest 6 bp stem binding footprint of bPNA+ makes the hybrid stem replacement method practical for noncovalent installation of synthetic probes of RNA interactions. We anticipate that bPNA+ structural probes will be useful for the study of tertiary interactions in long noncoding RNAs.

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA-RNA triplex formation. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dsRNA-binding PNAs (dbPNAs) can recognize internal Watson-Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases.

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The biochemistry and structural biology of the hammerhead ribozyme (HHR) have been well elucidated. The secondary and tertiary structural elements that enable sugar-phosphate bond scission to be catalyzed by this RNA are clearly understood. We have taken advantage of this knowledge base to test the extent to which synthetic molecules, may be used to trigger structure in secondary structure and tertiary interactions and thereby control HHR catalysis. These molecules belong to a family of molecules we generally call "bPNAs" based on our work on bifacial peptide nucleic acid (bPNA). This family of molecules displays the "bifacial" heterocycle melamine, which acts as a base-triple upon capturing two equivalents of thymine or uracil. Loosely structured internal oligouridylate bulges of 4-20 nucleotides can be restructured as triplex hybrid stems upon binding bPNAs. As such, a duplex stem element can be replaced with a bPNA triplex hybrid stem; similarly, a tertiary loop-stem interaction can be replaced with a loop-bPNA-stem complex. The ability to control RNA structure-function facilitates elucidation of these critical aspects of RNA recognition. In this chapter, we discuss how bPNAs are prepared and applied to study structure-function turn on in the hammerhead ribozyme system., (© 2019 Elsevier Inc. All rights reserved.)

Displaying ligands in a succinct and predictable manner is essential for elucidating multivalent molecular-level binding events. Organizing ligands with high precision and accuracy provides a distinct advantage over other ligand-display systems, such as polymers, because the number and position of the ligand(s) can be accurately and fully characterized. Here we describe the synthesis of peptide nucleic acids (PNAs), which are oligonucleotide mimics with a pseudopeptide backbone that can hybridize to oligonucleotides through Watson-Crick base pair to form highly predictable and organized scaffold for organizing a ligand. The ligand(s) are covalently attached to the PNA through a squarate coupling reaction that occurs between a free amine on the ligand and a free amine appended to the pseudopeptide backbone of the PNA. In this chapter we describe the synthesis of a L KγT monomer, which ultimately yields the free amine off the backbone of the PNA, incorporation of the monomer in a PNA oligomer, and the sequential squarate coupling to conjugate the ligand.

An efficient synthesis of Fmoc-protected ( S, S)- trans-cyclopentane PNA ( tcypPNA) monomers starting from mono-Boc-protected ( S, S)-1,2-cyclopentanediamine is reported. A general synthetic strategy was developed so that tcypPNA monomers with each nucleobase can be made in sufficient quantity and purity for use in solid-phase peptide synthesis (SPPS). The newly synthesized monomers were then successfully incorporated into 10-residue PNA oligomers using standard Fmoc chemistry for SPPS. The different tcypPNAs allow different positions in the sequence to be conformationally constrained with ( S, S)- trans-cyclopentane to determine the effects on binding to complementary DNA.

A combination of antibacterial agents should make the emergence of resistance in bacteria less probable. Thus we have analyzed the synergistic effects between antibacterial antisense peptide nucleic acids (PNA) and conventional antibiotics against Escherichia coli AS19 (lipopolysaccharide defective) strain and a derivative of a pathogenic strain E. coli O157:H7. PNAs were designed to target mRNA transcripts encoding the essential acyl carrier protein (gene acpP) and conjugated to the cell-penetrating peptide (KFF) 3 K for cellular uptake. Antibiotics included aminoglycosides, aminopenicillins, polymyxins, rifamycins, sulfonamides and trimethoprim. Synergies were evaluated using the checkerboard technique. Fractional Inhibitory Concentration indices (FICi) were calculated for all combinations based on the minimal inhibitory concentration of each individual agent. The results demonstrate two novel synergistic combinations of antimicrobial agents, namely, (KFF) 3 K-PNA anti-acpP with polymyxin B and (KFF) 3 K-PNA anti-acpP with trimethoprim (both with FICi = 0.38). Polymyxin B's synergy postulates cell wall targeted antibiotics as attractive agents to improve the uptake of PNA while trimethoprim's interaction with PNA my reveal a new inhibitory mechanism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid (MNP@PNA) was synthesized for use as both a magnetic nano-support and a probe for specific adsorption with complementary deoxyribonucleic acid (DNA). MNP@PNA with the size ranging between 120 and 170 nm in diameter was prepared via a free radical polymerization of acrylic acid in the presence of acrylamide-grafted MNP to obtain negatively charged magnetic nanoclusters, followed by ionic adsorption with PNA. According to fluorescence spectrophotometry and gel electrophoresis, this MNP@PNA can differentiate between fully matched, single-base mismatched and fully mismatched synthetic DNAs tagged with different fluorophores. UV-vis spectrophotometry and gel electrophoresis indicated that MNP@PNA can be used for specific adsorption with real DNA (zein gene of maize) having complementary sequence with the PNA probe. This novel anionic MNP conjugated with the PNA probe might be potentially applicable for use as a magnetic support for DNA base discrimination and might be a promising tool for testing genetic modification., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. Although it is central to many biological processes, such an approach has rarely been applied to the in situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic-acid probe containing a C-terminal thioester group and an N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of an RNA target and self-deactivation in its absence. The work has implications for the development of millamolecular nucleic-acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report here the construction of 3-way and 4-way peptide nucleic acid (PNA) junctions as basic structural units for PNA nanostructuring. The incorporation of amino acid residues into PNA chains makes PNA nanostructures with more structural complexity and architectural flexibility possible, as exemplified by building 3-way PNA junctions with tunable nanopores. Given that PNA nanostructures have good thermal and enzymatic stabilities, they are expected to have broad potential applications in biosensing, drug delivery and bioengineering.

Template-directed macromolecule synthesis is a hallmark of living systems. Inspired by this natural process, several fundamentally novel mechanisms for template-directed assembly of nucleic acid analogues have been developed. Although these approaches have broad significance, including potential applications in biotechnology and implications for the origins of life, there are unresolved challenges in how to characterize in detail the complex assembly equilibria associated with dynamic templated reactions. Here we describe mechanistic studies of template-directed dynamic assembly for thioester peptide nucleic acid (tPNA), an informational polymer that responds to selection pressures under enzyme-free conditions. To overcome some of the inherent challenges of mechanistic studies of dynamic oligomers, we designed, synthesized, and implemented tPNA-DNA conjugates. The DNA primer region affords a high level of control over the location and register of the tPNA backbone in relation to the template strand. We characterized the degree and kinetics of dynamic nucleobase mismatch correction at defined backbone positions. Furthermore, we report the fidelity of dynamic assembly in tPNA as a function of position along the peptide backbone. Finally, we present theoretical studies that explore the level of fidelity that can be expected for an oligomer having a given hybridization affinity in dynamic templated reactions and provide guidance for the future development of sequence self-editing polymers and materials. As our results demonstrate, the use of molecular conjugates of constitutionally static and dynamic polymers establishes a new methodology for expediting the characterization of the complex chemical equilibria that underlie the assembly of dynamic informational polymers.

Peptide Nucleic Acids (PNAs) are oligonucleotide mimics that can be used to block the biological action of microRNA, thus affecting gene expression post-transcriptionally. PNAs are obtained with solid-phase peptide synthesis, and can be easily conjugated to other peptides. Conjugation with R8-Peptide or modification of the PNA backbone (at C5 or C2 carbon) with arginine side chains allows efficient cellular uptake. The present protocol describes the synthesis of cationic PNAs that can be used alone as drugs or for efficient co-delivery in suitable inorganic nanocarriers.

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

A combination of fluorophore and nucleobase through a π-conjugated rigid linker integrates the base pairing and the fluorescence change into a single event. Such base discriminating fluorophore can change its fluorescence as a direct response to the base pairing event and therefore have advantages over tethered labels or base surrogates lacking the hydrogen-bonding ability. 8-(Pyrene-1-yl)ethynyl-adenine (A PyE ) has been extensively used as fluorescence labels in DNA and LNA, but it showed little discrimination between different nucleobases. Herein we investigated the synthesis, base pairing ability and optical properties of A PyE in pyrrolidinyl peptide nucleic acid - a DNA mimic that shows much stronger affinity and specificity towards DNA than natural oligonucleotides. The A PyE in PNA pairs specifically with thymine in the DNA strand, and resulted in 1.5-5.2-fold enhanced and blue-shifted fluorescence emission. Fluorescence quenching was observed in the presence of mismatched base or abasic site directly opposite to the A PyE . The behavior of A PyE in acpcPNA is distinctively different from DNA whereby a fluorescence was increased selectively upon duplex formation with complementary DNA and therefore emphasizing the unique advantages of using PNA as alternative oligonucleotide probes. Applications as color-shifting probe for detection of trinucleotide repeats in DNA were demonstrated, and the performance of the probe was further improved by combination with reduced graphene oxide as an external nanoquencher., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Self-assembled peptide-based hydrogels are emerging materials that have been exploited for wound healing, drug delivery, tissue engineering, and other applications. In comparison to synthetic polymer hydrogels, supramolecular peptide-based gels have advantages in biocompatibility, biodegradability, and ease of synthesis and modification. Modification of the emergent viscoelasticity of peptide hydrogels in a stimulus responsive fashion is a longstanding goal in the development of next-generation materials. In an effort to selectively modulate hydrogel viscoelasticity, we report herein a method to enhance the elasticity of β-sheet peptide hydrogels using specific molecular recognition events between functionalized hydrogel fibrils and biomolecules. Two distinct biomolecular recognition strategies are demonstrated: oligonucleotide Watson-Crick duplex formation between peptide nucleic acid (PNA) modified fibrils with a bridging oligonucleotide and protein-ligand recognition between mannose modified fibrils with concanavalin A. These methods to modulate hydrogel elasticity should be broadly adaptable in the context of these materials to a wide variety of molecular recognition partners.

Peptide nucleic acids (PNAs) derivatized with functional molecules are increasingly used in diverse biosupramolecular applications. PNAs have proven to be highly tolerant to modifications and different applications benefit from the use of modified PNAs, in particular modifications at the γ position. Herein we report simple protocols to access modified PNAs from iterative Ugi couplings which allow modular modifications at the α, β or γ position of the PNA backbone from simple starting materials. We demonstrate the utility of the method with the synthesis of several bioactive small molecules (a peptide ligand, a kinase inhibitor and a glycan)-PNA conjugates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose-phosphate was replaced by a peptide-like backbone. The absence of negative charge in the PNA backbone leads to several unique behaviors including a stronger binding and salt independency of the PNA-DNA duplex stability. However, PNA possesses poor aqueous solubility and cannot directly penetrate cell membranes. These are major obstacles that limit in vivo applications of PNA. In previous strategies, the PNA can be conjugated to macromolecular carriers or modified with positively charged side chains such as guanidinium groups to improve the aqueous solubility and cell permeability. In general, a preformed modified PNA monomer was required. In this study, a new approach for post-synthetic modification of PNA backbone with one or more hydrophilic groups was proposed. The PNA used in this study was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic acid dipeptide backbone (acpcPNA) that shows several advantages over the conventional PNA. The aldehyde modifiers carrying different linkers (alkylene and oligo(ethylene glycol)) and end groups (-OH, -NH 2 , and guanidinium) were synthesized and attached to the backbone of modified acpcPNA by reductive alkylation. The hybrids between the modified acpcPNAs and DNA exhibited comparable or superior thermal stability with base-pairing specificity similar to those of unmodified acpcPNA. Moreover, the modified apcPNAs also showed the improvement of aqueous solubility (10-20 folds compared to unmodified PNA) and readily penetrate cell membranes without requiring any special delivery agents. This study not only demonstrates the practicality of the proposed post-synthetic modification approach for PNA modification, which could be readily applied to other systems, but also opens up opportunities for using pyrrolidinyl PNA in various applications such as intracellular RNA sensing, specific gene detection, and antisense and antigene therapy.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

Here we report the synthesis of new PNA monomers for pseudocomplementary PNA (pcPNA) that are fully compatible with standard Fmoc chemistry. The thiocarbonyl group of the 2-thiouracil (sU) monomer was protected with the 4-methoxy-2-methybenzyl group (MMPM), while the exocyclic amino groups of diaminopurine (D) were protected with Boc groups. The newly synthesized monomers were incorporated into a 10-mer PNA oligomer using standard Fmoc chemistry for solid-phase synthesis. Oligomerization proceeded smoothly and the HPLC and MALDI-TOF MS analyses indicated that there was no remaining MMPM on the sU nucleobase. The new PNA monomers reported here would facilitate a wide range of applications, such as antigene PNAs and DNA nanotechnologies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of 19 F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by 19 F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA) 2 /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The intrinsic drawbacks encountered in bioactive peptides in chemical biology and biomedical sciences have diverted research efforts to the development of sequence-specific peptidomimetics that are capable of mimicking the structure and function of peptides and proteins. Modifications in the backbone and/or the side chain of peptides have been explored to develop biomimetic molecular probes or drug leads for biologically important targets. To expand the family of oligomeric peptidomimetics to facilitate their further application, we recently developed a new class of peptidomimetics, AApeptides based on a chiral peptide nucleic acid backbone. AApeptides are resistant to proteolytic degradation and amenable to enormous chemical diversification. Moreover, they could mimic the primary structure of peptides and also fold into discrete secondary structure such as helices and turn-like structures. Furthermore, they have started to show promise in applications in material and biomedical sciences. Herein, we highlight the structural design and some function of AApeptides and present our perspective on their future development.

Peptide nucleic acid (PNA) is an oligonucleotide mimic that recognizes and binds to nucleic acids. The strong binding affinity of PNA to mRNA coupled with its high sequence specificity enable antisense PNA to selectively inhibit (i.e., knockdown) the protein synthesis of a target gene. This novel technology provides a powerful tool for Campylobacter studies because molecular techniques have been relatively less well-developed for this bacterium as compared to other pathogens, such as Escherichia coli and Salmonella. This chapter describes a protocol for PNA-mediated antisense inhibition of gene expression in Campylobacter jejuni.