RESUMO
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 Aß1-42 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 ß-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.
Assuntos
Ácidos Nucleicos Peptídicos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , Peptídeos/química , Peptídeos/síntese química , Fenilalanina/química , Fenilalanina/análogos & derivados , Dicroísmo Circular , Dipeptídeos/química , Dipeptídeos/síntese químicaRESUMO
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 PN as modified nucleobases for recognition of cytosine in PN*C-G triplets. We found that a 3-pyridazinyl nucleobase formed significantly more stable PN*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.
Assuntos
Citosina/química , Ácidos Nucleicos Peptídicos/síntese química , Purinas/química , Piridazinas/química , RNA de Cadeia Dupla/química , Sequência de Bases , Ligação de Hidrogênio , Conformação Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Receptores ErbB/metabolismo , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/química , Receptor de Endotelina B/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Receptores ErbB/química , Receptores ErbB/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Receptor de Endotelina B/química , Receptor de Endotelina B/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
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.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Ácidos Nucleicos Peptídicos/análise , Ácidos Nucleicos Peptídicos/química , Pontos Quânticos/química , RNA/análise , RNA/química , Soluções Tampão , Catálise , Química Click , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico/métodos , Ácidos Nucleicos Peptídicos/síntese química , Técnicas de Síntese em Fase Sólida , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Corantes Fluorescentes , Imagem Óptica , Ácidos Nucleicos Peptídicos , Espectroscopia de Luz Próxima ao Infravermelho , Imagem Corporal Total , Animais , Linhagem Celular Tumoral , Análise de Dados , Imageamento Tridimensional , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/químicaRESUMO
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.
Assuntos
Marcação de Genes , Ácidos Nucleicos Peptídicos/administração & dosagem , Proteínas Recombinantes de Fusão , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Expressão Gênica , Humanos , Marcação por Isótopo , Imagem Molecular , Sondas Moleculares , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Técnicas de Síntese em Fase SólidaRESUMO
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-Zr4+-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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Detecção Precoce de Câncer , Corantes Fluorescentes/química , Neoplasias Pulmonares/diagnóstico por imagem , Ácidos Nucleicos Peptídicos/química , Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , DNA de Neoplasias/sangue , Corantes Fluorescentes/síntese química , Humanos , Queratina-19/sangue , Limite de Detecção , Estrutura Molecular , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , PolimerizaçãoRESUMO
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.
Assuntos
Proliferação de Células , MicroRNAs/genética , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Ácidos Nucleicos Peptídicos/farmacologia , RNA Mensageiro/antagonistas & inibidores , Química Computacional , Humanos , Microscopia Confocal , Simulação de Dinâmica Molecular , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Ácidos Nucleicos Peptídicos/síntese química , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
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 (K2M) 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-K2M 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.
Assuntos
DNA/química , Ácidos Nucleicos Peptídicos/síntese química , RNA/química , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , Peptídeos , Ligação Proteica , Conformação ProteicaRESUMO
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 5.97â¯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.
Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Análise em Microsséries/métodos , Mutação , Ácidos Nucleicos Peptídicos/síntese química , Biomarcadores Tumorais/genética , Humanos , Ácidos Nucleicos Peptídicos/genética , Sensibilidade e Especificidade , Técnicas de Síntese em Fase SólidaRESUMO
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 typeâ 1 and other related neuromuscular and neurodegenerative disorders.
Assuntos
Ácidos Nucleicos Peptídicos/química , Sondas RNA/química , RNA/química , Códon , Cisteína/química , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/genética , Polimerização , RNA/genética , Sondas RNA/síntese química , Sondas RNA/genética , Temperatura de TransiçãoRESUMO
"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.
Assuntos
Cisteína/química , Sondas de DNA/química , Corantes Fluorescentes/química , Ácidos Nucleicos Peptídicos/síntese químicaRESUMO
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 ß-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.
Assuntos
Materiais Biocompatíveis/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Materiais Biocompatíveis/síntese química , Sistemas de Liberação de Medicamentos , Elasticidade , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Ácidos Nucleicos Peptídicos/síntese química , Peptídeos/síntese química , Polímeros/síntese química , Polímeros/química , Engenharia TecidualRESUMO
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, ß 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.
Assuntos
Ácidos Nucleicos Peptídicos/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Glicoconjugados/síntese química , Glicoconjugados/química , Ligantes , Ácidos Nucleicos Peptídicos/química , Peptídeos/síntese química , Peptídeos/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Técnicas de Síntese em Fase Sólida/economiaRESUMO
The isoxazolidine ring represents one of the privileged structures in medicinal chemistry, and there have been an increasing number of studies on isoxazolidine and isoxazolidine-containing compounds. Optimization of the 1,3-dipolar cycloaddition (1,3-DC), original methods including electrophilic or palladium-mediated cyclization of unsaturated hydroxylamine, has been developed to obtain isoxazolidines. Novel reactions involving the isoxazolidine ring have been highlighted to accomplish total synthesis or to obtain bioactive compounds, one of the most significant examples being probably the thermic ring contraction applied to the total synthesis of (±)-Gelsemoxonine. The unique isoxazolidine scaffold also exhibits an impressive potential as a mimic of nucleosides, carbohydrates, PNA, amino acids, and steroid analogs. This review aims to be a comprehensive and general summary of the different isoxazolidine syntheses, their use as starting building blocks for the preparation of natural compounds, and their main biological activities.
Assuntos
Isoxazóis/química , Anti-Infecciosos/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Antineoplásicos/síntese química , Benzazepinas/síntese química , Carboidratos/síntese química , Ciclização , Reação de Cicloadição , Isoxazóis/síntese química , Nucleosídeos/síntese química , Oxazinas/síntese química , Oxirredução , Ácidos Nucleicos Peptídicos/síntese química , Peptidomiméticos/síntese química , Piridonas/síntese química , beta-Lactamas/síntese químicaRESUMO
Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.
Assuntos
Biomarcadores/sangue , Técnicas Biossensoriais , MicroRNA Circulante/sangue , Corantes Fluorescentes/química , Sondas de Ácido Nucleico/química , Ácidos Nucleicos Peptídicos/química , Neoplasias da Próstata/diagnóstico , MicroRNA Circulante/genética , MicroRNA Circulante/metabolismo , Cumarínicos/química , Humanos , Masculino , Hibridização de Ácido Nucleico , Sondas de Ácido Nucleico/metabolismo , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/síntese química , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.
Assuntos
Corantes Fluorescentes/química , Ácidos Nucleicos Peptídicos/química , Rodaminas/química , Linhagem Celular Tumoral , Citometria de Fluxo , Fluoresceínas/química , Fluoresceínas/efeitos da radiação , Corantes Fluorescentes/efeitos da radiação , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/efeitos da radiação , Fotodegradação , RNA de Cadeia Dupla/química , Rodaminas/efeitos da radiaçãoRESUMO
The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence.
Assuntos
Ácidos Nucleicos Peptídicos/metabolismo , Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Corantes Fluorescentes/química , Hidrólise , Hibridização de Ácido Nucleico , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/química , Peptídeos/síntese química , Peptídeos/química , Monoéster Fosfórico Hidrolases/química , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic-acid-based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment-sensitive 5-(benzofuran-2-yl)- and 5-(benzothiophen-2-yl)-uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene-modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn-on detection of G-quadruplex-forming promoter DNA sequences of human proto-oncogenes (c-myc and c-kit). Furthermore, the ability of benzothiophene-modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome-inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.
Assuntos
Fluorescência , Quadruplex G , Oncogenes/genética , Ácidos Nucleicos Peptídicos/análise , Ácidos Nucleicos Peptídicos/química , Proteínas Inativadoras de Ribossomos/metabolismo , Humanos , Estrutura Molecular , Ácidos Nucleicos Peptídicos/síntese químicaRESUMO
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.