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1.
Methods Mol Biol ; 2570: 119-128, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36156778

RESUMO

G-quadruplexes can be important as three-dimensional structures for aptamers as they improve the properties of the nucleic acids like higher nuclease degradation resistance. For the characterization of the quadruplex type and its formation, circular dichroism (CD) spectroscopy is one of the most common used identification methods. It is possible to differentiate the parallel and antiparallel G-quadruplex forms as well as the different number of nucleic acid strands by very specific CD spectral patterns at distinct absorption wavelengths. In this chapter, the protocol describes the model characterization of the anthracycline aptamer DRN-10 by CD spectroscopy, in order to show the usefulness and simple handling of this method for analyzing three-dimensional nucleic acid structures.


Assuntos
Aptâmeros de Nucleotídeos , Quadruplex G , Ácidos Nucleicos , Antraciclinas , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Conformação de Ácido Nucleico
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 284: 121758, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36029744

RESUMO

In consideration of relevance of antibiotic with food security, it is extremely desirable to propose sensitive and credible methods for antibiotic screening. Nevertheless, most of known approaches are developed based on fluorescence technique, which suffered from the interferences of background fluorescence and autoluminescence, and tedious labeling procedures, ascribing to the deficiency of high-performance and multifunctional dyes. Herein, we developed a novel iridium (III) complex (Ir-QAU)-based aptamer-promoted phosphorescence sensor for label-free, enzyme-free and highly sensitive detection of target antibiotic (kanamycin, Kan) based on target-switched hybridizing chain reaction (HCR). Ir-QAU was elaborately devised to present a signal-on response to G-quadruplex (G4) DNA against other DNAs due to its specific intercalation in G4 DNA and subsequent restriction of intra-molecular rotation. The recognition of H1 by Kan promoted the formation of Kan@H1 complexes, which hybridized with H2 and H3 via toehold-mediated hybridization reaction, subsequently switching HCR to produce large numbers of G4 DNA. Compared to Kan absence, abundant Ir-QAU was locked in G4 DNA to yield a significantly increased luminescence, which switches the luminescence analysis process of Kan with a limit of detection down to 0.38 pM. Furthermore, the Ir-QAU-based sensor was triumphantly applied to detect Kan in milk sample. We anticipate this work will disclose a new way to development of high-efficiency and practical luminescence sensor, and show a great potential for antibiotic-related food security.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Quadruplex G , Antibacterianos/análise , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Corantes , DNA , Irídio , Canamicina/análise , Limite de Detecção
3.
BMC Biol ; 20(1): 257, 2022 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-36372875

RESUMO

BACKGROUND: RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue. RESULTS: We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance. CONCLUSIONS: rG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.


Assuntos
Quadruplex G , Humanos , RNA/química , Transcriptoma , Células HEK293 , Análise de Sequência de RNA/métodos
4.
Biomacromolecules ; 23(11): 4795-4803, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36322676

RESUMO

Single-molecule methods offer high sensitivities with precisions superior to bulk assays. However, these methods are low in throughput and cannot repetitively interrogate the same cluster of molecular units. In this work, we investigate a tandem array of G-quadruplexes on a single-molecule DNA template with a throughput of at least two orders of magnitude higher than single-molecule force spectroscopy. During mechanical unfolding by optical tweezers, the array of G-quadruplexes experiences identical force, temperature, and ionic conditions, which not only reduce environmental noise but also render unfolding transitions indistinguishable among individual G-quadruplexes. The resultant ensemble behaviors are analyzed by scanning force diagrams, which reveals accurate F1/2 values, where 50% of G-quadruplexes are unfolded. Independent of the number of G-quadruplexes (n > 15) contained in a cluster, F1/2 can effectively evaluate G-quadruplex ligands in a new method called differential scanning forcemetry. When the same G-quadruplex cluster is subject to a series of constant forces in force-jump experiments, unfolding rate constants of G-quadruplexes can be effectively evaluated as a function of force. The high precision demonstrated in all of these measurements reflects the power of repetitive sampling on the same cluster of single-molecule entities under identical conditions. Since biomolecules such as DNA, RNA, and proteins can be conveniently incorporated in a tandem array, we anticipate that this ensemble assay on single-molecule entities (EASE) provides a generic means of ensemble force spectroscopy to amalgamate the accuracy of ensemble measurements with the precision of single-molecule methods.


Assuntos
Quadruplex G , Análise Espectral , Pinças Ópticas , Nanotecnologia , DNA/química
5.
Int J Mol Sci ; 23(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36361696

RESUMO

MST1R (RON) is a receptor of the MET tyrosine kinase receptor family involved in several cancers such as pancreas, breast, ovary, colon, and stomach. Some studies have shown that overexpression of MST1R increases the migratory and invasive properties of cancer cells. The promoter region of the oncogene MST1R is enriched in guanine residues that can potentially form G-quadruplexes (G4s), as it was observed in other oncogenic promoters such as KRAS and c-MYC. There is abundant literature that links the presence of G4s in promoter regions of oncogenes to diverse gene regulation processes that are not well understood. In this work, we have studied the reverse and forward sequence of MST1R promoter region using the G4Hunter software and performed biophysical studies to characterize the best scored sequences.


Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Guanina/química , Receptores Proteína Tirosina Quinases/metabolismo , Proto-Oncogenes
6.
Int J Mol Sci ; 23(21)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36362076

RESUMO

Taking a DNA sequence, a word with letters/bases A, T, G and C, as the relation between the generators of an infinite group π, one can discriminate between two important families: (i) the cardinality structure for conjugacy classes of subgroups of π is that of a free group on one to four bases, and the DNA word, viewed as a substitution sequence, is aperiodic; (ii) the cardinality structure for conjugacy classes of subgroups of π is not that of a free group, the sequence is generally not aperiodic and topological properties of π have to be determined differently. The two cases rely on DNA conformations such as A-DNA, B-DNA, Z-DNA, G-quadruplexes, etc. We found a few salient results: Z-DNA, when involved in transcription, replication and regulation in a healthy situation, implies (i). The sequence of telomeric repeats comprising three distinct bases most of the time satisfies (i). For two-base sequences in the free case (i) or non-free case (ii), the topology of π may be found in terms of the SL(2,C) character variety of π and the attached algebraic surfaces. The linking of two unknotted curves-the Hopf link-may occur in the topology of π in cases of biological importance, in telomeres, G-quadruplexes, hairpins and junctions, a feature that we already found in the context of models of topological quantum computing. For three- and four-base sequences, other knotting configurations are noticed and a building block of the topology is the four-punctured sphere. Our methods have the potential to discriminate between potential diseases associated to the sequences.


Assuntos
DNA Forma Z , Quadruplex G , Humanos , Sequência de Bases , Metodologias Computacionais , Teoria Quântica , Conformação de Ácido Nucleico , Telômero/genética
7.
Molecules ; 27(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36364401

RESUMO

G-quadruplexes (GQs) have become valid targets for anticancer studies in recent decades due to their multifaceted biological function. Herewith, we aim to quantify interactions of potential heterocyclic ligands (Ls) with model GQs. For seven 4-aminoquinazolines and three 2-heteroaryl perimidines, seven of this ten-membered group so far unknown, we use routine quantum chemical modeling. As shown in the literature, a preferred mode of interaction of heterocycles with cellular structures is stacking to exposable faces of G-quadruplexes. To exploit the energy of this interaction as a molecular descriptor and achieve the necessary chemical precision, we use state of the art large-scale density functional theory (DFT) calculations of stacked heterocycles to a GQ. Actually, the GQ has been simplified for the computation by stripping it off all pentose phosphate residues into a naked model of stacked guanine quartets. The described model thus becomes computable. The obtained heterocyclic ligand GQ.L stacking energies, that is, their GQ affinities, are the necessary ligand descriptors. Using the ligand biological inhibitory activities (IC50) on a human malignant melanoma A375 cell line, we obtain a good linear relationship between computed ligand stacking affinities to GQ, and experimental log (IC50) values. Based on the latter relationship, we discuss a putative mechanism of anticancer activity of heterocyclic ligands via stacking interactions with GQs and thereby controlling cell regulatory activity. This mechanism may tentatively be applied to other condensed five- and six-membered small heterocycles as well.


Assuntos
Antineoplásicos , Quadruplex G , Humanos , Ligantes , Relação Quantitativa Estrutura-Atividade , Antineoplásicos/química
8.
Chem Commun (Camb) ; 58(93): 12931-12934, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36321741

RESUMO

We constructed a minimum liquid-liquid phase separation model system to form liquid droplets using only G-quadruplex-forming oligonucleotides and R- and G-rich oligopeptides. We found that the G-quadruplex structure is an essential component for RNA to form droplets with the peptide. Based on this model system and our findings, droplet redissolution via structure transition from a G-quadruplex to a duplex was achieved in a sequence-specific manner.


Assuntos
Quadruplex G , Dicroísmo Circular , Oligonucleotídeos/química , RNA
9.
Viruses ; 14(11)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36423103

RESUMO

The integration of the HIV-1 genome into the host genome is an essential step in the life cycle of the virus and it plays a critical role in the expression, long-term persistence, and reactivation of HIV expression. To better understand the local genomic environment surrounding HIV-1 proviruses, we assessed the influence of non-canonical B-form DNA (non-B DNA) on the HIV-1 integration site selection. We showed that productively and latently infected cells exhibit different integration site biases towards non-B DNA motifs. We identified a correlation between the integration sites of the latent proviruses and non-B DNA features known to potently influence gene expression (e.g., cruciform, guanine-quadruplex (G4), triplex, and Z-DNA). The reactivation potential of latent proviruses with latency reversal agents also correlated with their proximity to specific non-B DNA motifs. The perturbation of G4 structures in vitro using G4 structure-destabilizing or -stabilizing ligands resulted in a significant reduction in integration within 100 base pairs of G4 motifs. The stabilization of G4 structures increased the integration within 300-500 base pairs from G4 motifs, increased integration near transcription start sites, and increased the proportion of latently infected cells. Moreover, we showed that host lens epithelium-derived growth factor (LEDGF)/p75 and cleavage and polyadenylation specificity factor 6 (CPSF6) influenced the distribution of integration sites near several non-B DNA motifs, especially G4 DNA. Our findings identify non-B DNA motifs as important factors that influence productive and latent HIV-1 integration and the reactivation potential of latent proviruses.


Assuntos
DNA de Forma B , Quadruplex G , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , HIV-1/genética , Motivos de Nucleotídeos , Latência Viral , DNA , Provírus/genética
10.
J Comput Aided Mol Des ; 36(12): 851-866, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36318393

RESUMO

In this work, the ab initio fragment molecular orbital (FMO) method was applied to calculate and analyze the binding energy of two biscarbene-Au(I) derivatives, [Au(9-methylcaffein-8-ylidene)2]+ and [Au(1,3-dimethylbenzimidazol-2-ylidene)2]+, to the DNA G-Quadruplex structure. The FMO2 binding energy considers the ligand-receptor complex as well as the isolated forms of energy-minimum state of ligand and receptor, providing a better description of ligand-receptor affinity compared with simple pair interaction energies (PIE). Our results highlight important features of the binding process of biscarbene-Au(I) derivatives to DNA G-Quadruplex, indicating that the total deformation-polarization energy and desolvation penalty of the ligands are the main terms destabilizing the binding. The pair interaction energy decomposition analysis (PIEDA) between ligand and nucleobases suggest that the main interaction terms are electrostatic and charge-transfer energies supporting the hypothesis that Au(I) ion can be involved in π-cation interactions further stabilizing the ligand-receptor complex. Moreover, the presence of polar groups on the carbene ring, as C = O, can improve the charge-transfer interaction with K+ ion. These findings can be employed to design new powerful biscarbene-Au(I) DNA-G quadruplex binders as promising anticancer drugs. The procedure described in this work can be applied to investigate any ligand-receptor system and is particularly useful when the binding process is strongly characterized by polarization, charge-transfer and dispersion interactions, properly evaluated by ab initio methods.


Assuntos
Antineoplásicos , Quadruplex G , Ligantes , Ouro , Antineoplásicos/química , DNA
11.
Nat Commun ; 13(1): 6016, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224201

RESUMO

KRAS is one of the most highly mutated oncoproteins, which is overexpressed in various human cancers and implicated in poor survival. The G-quadruplex formed in KRAS oncogene promoter (KRAS-G4) is a transcriptional modulator and amenable to small molecule targeting. However, no available KRAS-G4-ligand complex structure has yet been determined, which seriously hinders the structure-based rational design of KRAS-G4 targeting drugs. In this study, we report the NMR solution structures of a bulge-containing KRAS-G4 bound to berberine and coptisine, respectively. The determined complex structure shows a 2:1 binding stoichiometry with each compound recruiting the adjacent flacking adenine residue to form a "quasi-triad plane" that stacks over the two external G-tetrads. The binding involves both π-stacking and electrostatic interactions. Moreover, berberine and coptisine significantly lowered the KRAS mRNA levels in cancer cells. Our study thus provides molecular details of ligand interactions with KRAS-G4 and is beneficial for the design of specific KRAS-G4-interactive drugs.


Assuntos
Berberina , Quadruplex G , Adenina , Berberina/análogos & derivados , Berberina/farmacologia , Genes ras , Humanos , Ligantes , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Mensageiro
12.
Biochemistry ; 61(19): 2073-2087, 2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36193632

RESUMO

During its life cycle, the predatory bacterium Bdellovibrio bacteriovorus switches between an attack and a growth phase, each of which is characterized by a distinct pattern of gene expression. Twenty-one potential G-quadruplex-forming sequences (PQFS) have been identified in the Bdellovibrio genome. These G-rich sequences are prevalent within open reading frames and nearly evenly distributed between the template and the coding strand, suggesting that they could play a role in gene expression and life cycle switching. Published transcriptomic data show that the genes nearest these sequences are not (de)activated together during the same phases of the life cycle. We explored the biophysical properties of three identified PQFS using circular dichroism (CD) spectroscopy and gel electrophoresis and demonstrated that all three sequences fold into stable unimolecular quadruplexes with distinct topologies. In the presence of their complementary strands, each forms an equilibrium mixture of duplex and quadruplex in which quadruplex formation is favored at higher temperatures. Once the quadruplexes are folded, they are slow to form a duplex when the complementary strand is added, with one sequence requiring the equivalent of many Bdellovibrio lifetimes to do so. Using a variety of cosolutes, we showed that molecular crowding mimicking cellular conditions stabilizes the quadruplex structures and induces structural transitions to the parallel topology regardless of the original topology. Taken together, these experiments suggest that Bdellovibrio PQFS are capable of forming quadruplexes in vivo and thereby playing a role in gene expression.


Assuntos
Bdellovibrio bacteriovorus , Quadruplex G , Dicroísmo Circular , DNA/química , Replicação do DNA
13.
Anal Chim Acta ; 1230: 340393, 2022 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-36192064

RESUMO

The concentration variation of phenylalanine (Phe), an essential amino acid in humans, can cause metabolism disorders and even mental disability. Sensitive and convenient monitoring of Phe is therefore important for disease diagnosis. We describe here the establishment of a new aptamer-based, sensitive and label-free colorimetric Phe detection strategy by integrating catalytic hairpin assembly (CHA) and Mg2+-dependent DNAzyme amplification cascades. The target Phe coordinates with pentamethylcyclopentadienyl rhodium(III) chloride dimer [(Cp*RhCl2)2] to form a complex that has a high affinity to the corresponding aptamer sequence. Upon its binding to the aptamers in DNA duplex probes, ssDNA strands are released to trigger subsequent CHA reactions for the formation of many DNAzymes, which cleave the substrate signal probes to liberate lots of CHA initiation strands and free G-quadruplexes to realize the cascaded amplifications. Hemin further associates with the many G-quadruplexes to yield hemin/G-quadruplex mimicking peroxidases, which catalyze solution of substrate to exhibit highly enhanced UV-vis adsorption for detecting Phe at 0.19 µM level. At the meantime, the monitoring of Phe in diluted serums with high selectivity has also been demonstrated by the developed method, indicating its potential for simple diagnosis of Phe-related diseases.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , Ródio , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Cloretos , Colorimetria/métodos , Sondas de DNA/química , DNA Catalítico/química , Hemina/química , Humanos , Peroxidases/metabolismo , Fenilalanina
14.
Int J Mol Sci ; 23(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36232294

RESUMO

The initial aim of this work was to elucidate the mutual influence of different single-stranded segments (loops and caps) on the thermodynamic stability of RNA G-quadruplexes. To this end, we used a new NAB-GQ-builder software program, to construct dozens of two-tetrad G-quadruplex topologies, based on a designed library of sequences. Then, to probe the sequence-morphology-stability relationships of the designed topologies, we performed molecular dynamics simulations. Their results provide guidance for the design of G-quadruplexes with balanced structures, and in turn programmable physicochemical properties for applications as biomaterials. Moreover, by comparative examinations of the single-stranded segments of three oncogene promoter G-quadruplexes, we assess their druggability potential for future therapeutic strategies. Finally, on the basis of a thorough analysis at the quantum mechanical level of theory on a series of guanine assemblies, we demonstrate how a valence tautomerism, triggered by a coordination of cations, initiates the process of G-quadruplex folding, and we propose a sequential folding mechanism, otherwise dictated by the cancellation of the dipole moments on guanines.


Assuntos
Quadruplex G , Materiais Biocompatíveis , Cátions/química , Eletrônica , Guanina/química
15.
Biochim Biophys Acta Gen Subj ; 1866(12): 130252, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36216170

RESUMO

BACKGROUND: Non-B DNA conformations are molecular structures that do not follow the canonical DNA double helix. Mutagenetic instability in nuclear and mitochondrial DNA (mtDNA) genomes has been associated with simple non-B DNA conformations, as hairpins or more complex structures, as G-quadruplexes. One of these structures is Structure A, a cloverleaf-like non-B conformation predicted for a 93-nt (nucleotide) stretch of the mtDNA control region 5'-peripheral domain. Structure A is embedded in a hot spot for the 3' end of human mtDNA deletions revealing its importance in influencing the mutational instability of the mtDNA genome. METHODS: To better characterize Structure A, we predicted its 3D conformation using state-of-art methods and algorithms. The methodologic workflow consisted in the prediction of non-B conformations using molecular dynamics simulations. The conservation scores of alignments of the Structure A region in humans, primates, and mammals, was also calculated. RESULTS: Our results show that these computational methods are able to measure the stability of non-B conformations by using the level of base pairing during molecular dynamics. Structure A showed high stability and low flexibility correlated with high conservation scores in mammalian, more specifically in primate lineages. CONCLUSIONS: We showed that 3D non-B conformations can be predicted and characterized by our methodology. This allowed the in-depth analysis of the structure A, and the main results showed the structure remains stable during the simulations. GENERAL SIGNIFICANCE: The fine-scale atomic molecular determination of this type of non-B conformation opens the way to perform computational molecular studies that can show their involvement in mtDNA cellular mechanisms.


Assuntos
Quadruplex G , Simulação de Dinâmica Molecular , Animais , Humanos , Conformação de Ácido Nucleico , DNA Mitocondrial/genética , DNA Mitocondrial/química , Pareamento de Bases , Mamíferos
16.
Biochemistry ; 61(21): 2390-2397, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36260746

RESUMO

Oxidation of a guanine nucleotide in DNA yields an 8-oxoguanine nucleotide (oxoG) and is a mutagenic event in the genome. Due to different arrangements of hydrogen-bond donors and acceptors, oxoG can affect the secondary structure of nucleic acids. We have investigated base pairing preferences of oxoG in the core of a tetrahelical G-quadruplex structure, adopted by analogues of d(TG4T). Using spectroscopic methods, we have shown that G-quartets can be fully substituted with oxoG nucleobases to form an oxoG-quartet with a revamped hydrogen-bonding scheme. While an oxoG-quartet can be incorporated into the G-quadruplex core without distorting the phosphodiester backbone, larger dimensions of the central cavity change the cation localization and exchange properties.


Assuntos
Quadruplex G , Guanina , Guanina/química , DNA/química , Hidrogênio , Conformação de Ácido Nucleico
17.
Molecules ; 27(20)2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36296374

RESUMO

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.


Assuntos
Aptâmeros de Nucleotídeos , Carcinoma de Células Escamosas , Quadruplex G , Neoplasias da Língua , Humanos , Ligantes , Aptâmeros de Nucleotídeos/química
18.
Bioorg Med Chem ; 73: 116971, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36208542

RESUMO

G-quadruplex (G4) structures are non-canonical DNA/RNA secondary structures able to form within guanine rich nucleic acids sequences. They are present in several regions of the human genome including gene promoters, untranslated sequences, and telomeres. Due to their biological relevance G4 structures are considered important drug targets, in particular for anticancer therapies, leading to the development of G4 stabilizing small molecules. Telomeric regions have received special attention in this field since they can fold into several distinct intramolecular G-quadruplexes topologies. Herein, we report the synthesis of 2,9-disubstituted-1,10-phenanthroline derivatives and their ability to stabilize different intramolecular telomeric G4 sequences. We evaluated ligand-induced stabilization, selectivity and specificity of ligands using Förster Resonance Energy Transfer (FRET) melting experiments and circular dichroism (CD). In addition, we assessed the cytotoxicity of ligands against two cancer cell lines (A549 and H1299) and one healthy cell line (NHDF).


Assuntos
Quadruplex G , Dicroísmo Circular , DNA/química , Guanina , Humanos , Ligantes , Fenantrolinas , RNA , Telômero
19.
J Vis Exp ; (188)2022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36282711

RESUMO

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 µM theophylline in as little as 15 min.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , RNA Catalítico , DNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/metabolismo , Teofilina , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Oligonucleotídeos , Peroxidases , Aptâmeros de Nucleotídeos/química
20.
Anal Chim Acta ; 1233: 340515, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36283789

RESUMO

Sensitive and selective detection of neutrophil gelatinase-associated lipocalin (NGAL) is critical for the prediction and early diagnosis of acute renal injury. In this work, the establishment of an aptamer-based, highly sensitive and label-free method for detecting NGAL in diluted human serums via metal ion-dependent DNAzyme- and exonuclease III (Exo III)-triggered recycling signal amplification cascades is described. NGAL binds with the aptamer strands in the DNAzyme/aptamer duplexes and results in the liberation of the metal ion-dependent DNAzyme sequences to cleave the hairpin signal probes on the electrode to liberate the G-quadruplex and intermediate strands. The released intermediate strands further complement with the DNAzyme/aptamer duplexes to form favorable substrate for Exo III, which digests the duplexes to release the DNAzyme strands to initiate the cascaded recycling cycles for the yield of plenty of G-quadruplex strands. Hemin can associate with G-quadruplex strands to produce many G-quadruplex/hemin complexes and electrochemical reduction of hemin thus generates highly amplified current for detecting NGAL with the detection limit of 4.45 ng mL-1. Such biosensor also shows high selectivity and can be utilized for monitoring NGAL spiked in diluted serum, indicating its extension potential for detecting various protein biomarkers with different aptamers for disease diagnosis.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , Humanos , DNA Catalítico/química , Hemina/química , Lipocalina-2/metabolismo , Técnicas Biossensoriais/métodos , Limite de Detecção , Técnicas Eletroquímicas/métodos , Aptâmeros de Nucleotídeos/química
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