Peptide Nucleic Acid Conjugates Research Articles

Evaluating delivery of peptide nucleic acids to Gram-negative bacteria using differently linked membrane-active peptides and their stapled analogs

Antisense oligonucleotides have been developed as therapeutic compounds, with peptide nucleic acid (PNA) emerging as a promising nucleic acid mimic for antimicrobial applications. To be effective, PNAs must be internalized into bacterial cells, as they are not naturally absorbed. A strategy to improve PNA membrane penetration and cellular uptake involves covalently conjugating them to cell-penetrating peptides. However, these membrane-active peptides can exhibit cytotoxicity, and their efficiency as PNA carriers needs to be enhanced. Therefore, we explored new peptide–PNA conjugates and their linkers to understand how they affect PNA uptake into bacteria. We conjugated PNA to two peptides, anoplin and (KFF)3K, along with their structurally stabilized hydrocarbon-stapled derivatives, and evaluated their transport into various bacterial strains. The PNA sequence targeted bacterial mRNA encoding the essential acyl carrier protein. As linkages, we used either a non-cleavable 8-amino-2,6-dioxaoctanoyl (ethylene glycol, eg1) linker or a reducible disulfide bridge. We found that the hydrocarbon-stapled peptides did not enhance PNA delivery, despite the strong inner- and outer-membrane-penetrating capabilities of the standalone peptides. Additionally, the disulfide bridge linkage, which is cleavable in the bacterial cytoplasm, decreased the antimicrobial activity of the peptide–PNA conjugates. Notably, we identified anoplin as a new potent PNA carrier peptide, with the anoplin–eg1–PNA conjugate demonstrating antibacterial activity against E. coli and S. Typhimurium strains in the 2–4 µM range.

Diffusion of 1O2 along the PNA backbone diminishes the efficiency of photooxidation of PNA/DNA duplexes by biphenyl photosensitizer

Nitrobiphenyl photosensitizer (NBP)-peptide nucleic acids (PNA) conjugates were synthesized to develop a tool for photo-knockdown of target DNAs. The presence of NBP hardly hindered duplex formation with the complementary single strand DNA as demonstrated by the comparison of Tm values and CD spectra with those for standard PNA/DNA duplexes. However, the photooxidation of guanines in NBP-PNA/DNAs was significantly less effective than those of corresponding NBP-DNA/DNA. Production of singlet oxygen (1O2) during the photooxidation was confirmed by consumption of furfuryl alcohol, a 1O2 detector. The poor photooxidation efficiency was ameliorated with 1O2 generated from an externally added NBP derivative. It was found that, when complexed with the sticky end of a double strand DNA, NBP-PNA was able to photooxidize G in the DNA/DNA duplex region, whereas G in the PNA/DNA duplex region was considerably unreactive. These results suggest that 1O2 produced from NBP-PNA tends to quench during diffusion along the PNA/DNA backbone, whereas quenching is less likely during diffusion along DNA/DNA region.

Papain-Mediated Conjugation of Peptide Nucleic Acids to Delivery Peptides: A Density Functional Theory/Molecular Mechanics Metadynamics Study in Aqueous and Organic Solvent.

Enzymatic peptide synthesis is a powerful alternative to solid-phase methods, as enzymes can have high regio- and stereoselectivity and high yield and require mild reaction conditions. This is beneficial in formulation research due to the rise of nucleic acid therapies. Peptide nucleic acids (PNAs) have a high affinity toward DNA and RNA, and their solubility and cellular delivery can be improved via conjugation to peptides. Here, we designed and assessed the viability of the papain enzyme to conjugate four PNA-peptide models in water and an organic solvent using QM/MM metadynamics. We found that the reactions in water yield better results, where three conjugates could potentially be synthesized by the enzyme, with the first transition state as the rate-limiting step, with an associated energy of 14.53 kcal mol-1, although with a slight endergonic profile. The results highlight the importance of considering the enzyme pockets and different substrate acceptivities and contribute to developing greener, direct, and precise synthetic routes for nucleic acid-based therapies. By exploring the enzyme's potential in conjunction with chemical synthesis, current protocols can be simplified for the synthesis of longer nucleic acids and peptide sequences (and, by extension, proteins) from smaller oligo or peptide blocks.

Effective lowering of α-synuclein expression by targeting G-quadruplex structures within the SNCA gene

Alpha-synuclein, encoded by the SNCA gene, is a pivotal protein implicated in the pathogenesis of synucleinopathies, including Parkinson's disease. Current approaches for modulating alpha-synuclein levels involve antisense nucleotides, siRNAs, and small molecules targeting SNCA's 5′-UTR mRNA. Here, we propose a groundbreaking strategy targeting G-quadruplex structures to effectively modulate SNCA gene expression and lowering alpha-synuclein amount. Novel G-quadruplex sequences, identified on the SNCA gene's transcription starting site and 5′-UTR of SNCA mRNAs, were experimentally confirmed for their stability through biophysical assays and in vitro experiments on human genomic DNA. Biological validation in differentiated SH-SY5Y cells revealed that well-known G-quadruplex ligands remarkably stabilized these structures, inducing the modulation of SNCA mRNAs expression, and the effective decrease in alpha-synuclein amount. Besides, a novel peptide nucleic acid conjugate, designed to selectively disrupt of G-quadruplex within the SNCA gene promoter, caused a promising lowering of both SNCA mRNA and alpha-synuclein protein. Altogether our findings highlight G-quadruplexes' key role as intriguing biological targets in achieving a notable and successful reduction in alpha-synuclein expression, pointing to a novel approach against synucleinopathies.

Towards a templated reaction that translates RNA in cells into a proaptotic peptide-PNA conjugate.

Nucleic acid-templated chemistry opens the intriguing prospect of triggering the synthesis of drugs only in diseased cells. Herein, we explore the feasibility of using RNA-templated chemical reactions for the activation of a known Smac peptidomimetic compound (SMC), which has proapoptotic activity. Two peptide nucleic acid (PNA) conjugates were used to enable conditional activation of a masked SMC by reduction of an azide either by Staudinger reduction or catalytic photoreduction using a ruthenium complex. The latter provided ~135 nM SMC-PNA on as little as 10nM (0.01 eq.) template. For the evaluation of the templated azido-SMC reduction system in cellulo, a stable HEK 293 cell line was generated, which overexpressed a truncated, non-functional form of the XIAP mRNA target. We furthermore describe the development of electroporation protocols that enable a robust delivery of PNA conjugates into HEK 293 cells. The action of the reactive PNA conjugates was evaluated by viability and flow cytometric apoptosis assays. In addition, electroporated probes were re-isolated and analyzed by ultra-high performance liquid chromatography (UPLC). Unfortunately, the ruthenium-PNA conjugate proved phototoxic, and treatment of cells with PNA-linked reducing agent and the azido-masked SMC conjugate did not result in a greater viability loss than treatment with scrambled sequence controls. Intracellular product formation was not detectable. A control experiment in total cellular RNA isolate indicated that the templated reaction can in principle proceed in a complex system. The results of this first-of-its-kind study reveal the numerous hurdles that must be overcome if RNA molecules are to trigger the synthesis of pro-apoptotic drugs inside cells.

Multivalent Lactobionic Acid and N-Acetylgalactosamine-Conjugated Peptide Nucleic Acids for Efficient In Vivo Targeting of Hepatocytes.

Peptide nucleic acids (PNAs) are used/applied in various studies to target genomic DNA and RNA to modulate gene expression. Non-specific targeting and rapid elimination always remain a challenge for PNA-based applications. Here, the synthesis, characterization, in vitro and in vivo study of di lactobionic acid (diLBA) and tris N-acetyl galactosamine (tGalNAc) conjugated PNAs for liver-targeted delivery are reported. For proof of concept, diLBA, and tGalNAc conjugated PNAs (anti-miR-122 PNAs) were synthesized to target microRNA-122 (miR-122) which is over-expressed in the hepatic tissue. Different lengths of anti-miR-122 PNAs conjugated with diLBA and tGalNAc are tested. Cell culture and in vivo analyses to determine biodistribution, efficacy, and toxicity profile are performed. This work indicates that diLBA conjugates show significant retention in hepatocytes in addition to tGalNAc conjugates after in vivo delivery. Full-length PNA conjugates show significant downregulation of miR-122 levels and subsequent de-repression of its downstream targets with no evidence of toxicity. The results provide a robust framework for ligand-conjugated delivery systems for PNAs that can be explored for broader biomedical applications.

Kinetic analysis of highly effective triplex formation between a small molecule-peptide nucleic acid conjugate probe and the influenza A virus RNA promoter region at neutral pH.

In order to overcome the pH limitations of triplex-forming peptide nucleic acid (PNA) in binding to double-stranded RNA (dsRNA), we have recently proposed a new design of triplex-forming PNA-based fluorogenic probes that work at neutral pH for sensing the panhandle structure of the influenza A virus (IAV) RNA promoter region. Our strategy is based on the conjugation of a small molecule (DPQ) capable of selectively binding to the internal loop structure with the triplex-forming forced intercalation of thiazole orange (tFIT) probe with natural PNA nucleobases. In this work, the triplex formation of tFIT-DPQ conjugate probes with IAV target RNA at neutral pH was examined by means of a stopped-flow technique UV melting and fluorescence titration experiments. The obtained results revealed that (i) the conjugation strategy is responsible for the observed strong binding affinity due to a very fast association rate constant and a slow dissociation rate constant; (ii) the binding follows a pattern of the DPQ unit binding first to the internal loop region, followed by the tFIT unit binding to the complementary dsRNA region. Our results emphasize the importance of both the tFIT and the DPQ components of the conjugate probe design and revealed an association mechanism for the tFIT-DPQ probe-dsRNA triplex formation towards the IAV RNA at neutral pH.

Sulfonium-Driven Neoantigen-Released DNA Nanodevice as a Precise Vaccine for Tumor Immunotherapy and Prevention.

Peptide-based neoantigen vaccines hold tremendous potential for personalized tumor immunotherapy. However, effective delivery and controllable release of antigen peptides remain major challenges in stimulating robust and sustained immune responses. Programmable DNA nanodevices provide accurate fixed positions for antigens, which are convenient for the calculation of clinical dosage, and hold great potential as precise carriers. Here, a peptide-nucleic acid conjugate was prepared, which was driven by a propargyl sulfonium-based efficient and reversible bio-orthogonal reaction under weakly alkaline conditions, and folded into regular DNA nanodevice vaccines. The well-defined nanoplatform not only exhibits outstanding stability in serum, satisfactory safety, and effective internalization by antigen-presenting cells (RAW264.7 and BMDCs) but also obviously enhances cytokine (TNF-α, IL-6, and IL-12) secretion for further immune response. In vivo, the nanovaccine cooperating with OVA model antigens and CpG adjuvants stimulated an antigen-specific CD8+T cell response, significantly preventing the lung metastases of melanoma. In the B16-OVA tumor-bearing model, the growth inhibition rate of melanoma reached up to 50%. Similarly, the DNA nanodevice with neoantigen induced up to a maximum degree of complete MC-38 tumor regression in 80% of mice, possibly owing to antigen peptide reversible release driven by sulfonium and further cross-presentation. In brief, this study demonstrates that DNA nanodevices with sulfonium centers can provide a precise, biocompatible, and effective co-delivery vaccine platform for tumor immunotherapy and prevention.

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli.

Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting 11 essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e.dnaB, ftsZ and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that 9mer PNAs are generally as effective in inhibiting bacterial growth as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.

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.

Automated Flow Synthesis of Peptide-PNA Conjugates.

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5′ untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 μM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.

Direct and Efficient Conjugation of Quantum Dots to DNA Nanostructures with Peptide-PNA.

DNA nanotechnology has proven to be a powerful strategy for the bottom-up preparation of colloidal nanoparticle (NP) superstructures, enabling the coordination of multiple NPs with orientation and separation approaching nanometer precision. To do this, NPs are often conjugated with chemically modified, single-stranded (ss) DNA that can recognize complementary ssDNA on the DNA nanostructure. The limitation is that many NPs cannot be easily conjugated with ssDNA, and other conjugation strategies are expensive, inefficient, or reduce the specificity and/or precision with which NPs can be placed. As an alternative, the conjugation of nanoparticle-binding peptides and peptide nucleic acids (PNA) can produce peptide-PNA with distinct NP-binding and DNA-binding domains. Here, we demonstrate a simple application of this method to conjugate semiconductor quantum dots (QDs) directly to DNA nanostructures by means of a peptide-PNA with a six-histidine peptide motif that binds to the QD surface. With this method, we achieved greater than 90% capture efficiency for multiple QDs on a single DNA nanostructure while preserving both site specificity and precise spatial control of QD placement. Additionally, we investigated the effects of peptide-PNA charge on the efficacy of QD immobilization in suboptimal conditions. The results validate peptide-PNA as a viable alternative to ssDNA conjugation of NPs and warrant studies of other NP-binding peptides for peptide-PNA conjugation.

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation.

Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133–150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133–150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The in vitro analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.

Impact of Chirality and Position of Lysine Conjugation in Triplex-Forming Peptide Nucleic Acids.

Conjugation with cationic lysine residues improves the biophysical and biological properties of peptide nucleic acids (PNAs). A single lysine is routinely used to improve the solubility and prevent aggregation of the neutral and hydrophobic amide backbone of PNA. Literature precedents include the attachment of lysine at either the N- or the C-terminus. Moreover, conjugation with short lysine peptides (four to eight residues) improves the cellular uptake of PNA akin to more complex cell-penetrating peptides. Herein, we report a systematic study of the effect of lysine location (N- vs C-terminus) and chirality (d- vs l-) on triple-helical binding of PNA to double-stranded RNA and DNA (dsRNA and dsDNA). The results confirmed our earlier findings that conjugation with lysine significantly increased the stability of PNA-dsRNA and PNA-dsDNA triplexes and that PNA affinity for dsRNA was about an order of magnitude higher than for the same sequence of dsDNA. In contrast, conjugation of PNA with noncharged amino acids decreased the affinity of PNA. Surprisingly, neither the location nor the chirality of lysine had significant impact on PNA affinity for either dsRNA or dsDNA. The results are consistent with the lack of chiral preorganization of single-stranded PNAs, even after conjugation with four d- or l-amino acids. Instead, the positive charge of lysine appears to be the main driving force behind the increased affinity.

Coupling of an antifouling and reusable nanoplatform with catalytic hairpin assembly for highly sensitive detection of nucleic acids using zeta potential as signal readout

Zeta potential analyzer has been becoming a routine instrument for nanomaterial characterization, which allows simple, non-invasive, highly sensitive, and reagentless measurement. However, its application in quantitative bioanalysis is greatly hampered by the dearth of appropriate sensing strategy, high background signal of nanomaterials and severe interference in complex biofluids. Herein, we develop a highly sensitive method to detect nucleic acids based on an antifouling and reusable nanoplatform using zeta potential as signal readout. In this work, the nanoplatform, composed of a magnetic nanoparticle (MNP) core and an antifouling zwitterionic lipid membrane, not only resists on the nonspecific adsorption of proteins, but facilitates its separation from complex detection system, enabling accurate measurement of zeta potential. After conjugation of uncharged peptide nucleic acids, the resultant nanoconjugates can capture negatively charged target nucleic acids, significantly reducing interfacial zeta potential. The proposed method can detect picomolar analyte in a single incubation step or fetomolar analyte with the use of chain hairpin assembly. Additionally, the nanoplatform can be reused by mild nuclease treatment, which is rather difficult to be achieved by other nanoparticle-based detection methods. Overall, this method provides a new insight to use zeta potential as signal output for quantitative bioanalysis.

Ku80-Targeted pH-Sensitive Peptide-PNA Conjugates Are Tumor Selective and Sensitize Cancer Cells to Ionizing Radiation.

The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be achieved through the use of pH-low insertion peptides (pHLIP), which take advantage of the acidity of the tumor microenvironment to deliver cargoes selectively to tumor cells. We developed a pHLIP-peptide nucleic acid (PNA) conjugate as an antisense reagent to reduce expression of the otherwise undruggable DNA double-strand break repair factor, KU80, and thereby radiosensitize tumor cells. Increased antisense activity of the pHLIP-PNA conjugate was achieved by partial mini-PEG sidechain substitution of the PNA at the gamma position, designated pHLIP-αKu80(γ). We evaluated selective effects of pHLIP-αKu80(γ) in cancer cells in acidic culture conditions as well as in two subcutaneous mouse tumor models. Fluorescently labeled pHLIP-αKu80(γ) delivers specifically to acidic cancer cells and accumulates preferentially in tumors when injected i.v. in mice. Furthermore, pHLIP-αKu80(γ) selectively reduced KU80 expression in cells under acidic conditions and in tumors in vivo. When pHLIP-αKu80(γ) was administered to mice prior to local tumor irradiation, tumor growth was substantially reduced compared with radiation treatment alone. Furthermore, there was no evidence of acute toxicity associated with pHLIP-αKu80(γ) administration to the mice. These results establish pHLIP-αKu80(γ) as a tumor-selective radiosensitizing agent. IMPLICATIONS: This study describes a novel agent, pHLIP-αKu80(γ), which combines PNA antisense and pHLIP technologies to selectively reduce the expression of the DNA repair factor KU80 in tumors and confer tumor-selective radiosensitization.

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids.

Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

Selective targeting of mutually exclusive DNA G-quadruplexes: HIV-1 LTR as paradigmatic model.

Targeting of G-quadruplexes, non-canonical conformations that form in G-rich regions of nucleic acids, has been proposed as a novel therapeutic strategy toward several diseases, including cancer and infections. The unavailability of highly selective molecules targeting a G-quadruplex of choice has hampered relevant applications. Herein, we describe a novel approach, based on naphthalene diimide (NDI)-peptide nucleic acid (PNA) conjugates, taking advantage of the cooperative interaction of the NDI with the G-quadruplex structure and hybridization of the PNA with the flanking region upstream or downstream the targeted G-quadruplex. By biophysical and biomolecular assays, we show that the NDI-PNA conjugates are able to specifically recognize the G-quadruplex of choice within the HIV-1 LTR region, consisting of overlapping and therefore mutually exclusive G-quadruplexes. Additionally, the conjugates can induce and stabilize the least populated G-quadruplex at the expenses of the more stable ones. The general and straightforward design and synthesis, which readily apply to any G4 target of choice, together with both the red-fluorescent emission and the possibility to introduce cellular localization signals, make the novel conjugates available to selectively control G-quadruplex folding over a wide range of applications.

Synthesis and physicochemical properties of 20-mer peptide nucleic acid conjugates with testosterone 17β-carboxylic acid

Although peptide nucleic acids (PNAs) have improved nuclease resistance compared with DNA or RNA, it is difficult to synthesize long PNAs because of poor elongation yield. Herein we synthesized 20-mer PNAs (PNA20), targeting Nnmt mRNA, as well as its conjugate with testosterone 17β-carboxylic acid, in high purity and yield. This synthesis was conducted using Oxyma as a condensation agent and NMP as a solvent for Fmoc-PNA-C(Bhoc)-OH. The resistance of PNA20 to exonuclease was higher than that of RNA. Furthermore, the abilities of PNA20 and its conjugate to bind to complementary DNA were stronger than that of DNA or RNA. These findings lay the basis for the synthesis of long PNA derivatives toward oligonucleotide therapeutics.

Peptide Nucleic Acid Conjugates of Quinone Methide Precursors Alkylate Ribonucleic Acid after Activation with Light.

Quinone methide precursors 2 and 3 were protected with a photoreactive 2-nitrobenzyl group and conjugated to peptide nucleic acids (PNA) using a Huisgen click reaction. After brief irradiation at 365 nm, cross-linking with complementary RNA strands started and was analyzed with an ALFexpress sequencer. When this method was used, the gel temperature had a major influence on apparent rates. Quinone methides are known to form transient as well as stable bonds with nucleotides. Although both were detected at 25 °C, analysis at 57 °C only recorded the stable types of cross-links, suggesting much slower alkylation kinetics. Linker 11 allowed us to attach quinone methides to internal positions of the PNA/RNA duplex and to capture a model of miR-20a with good efficiency.