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1.
ACS Appl Mater Interfaces ; 13(33): 39711-39718, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34402304

RESUMO

The manufacture of DNA origami nanostructures with highly ordered functional motifs is of great significance for biomedical applications. Here, we present a robust strategy to produce customized scaffolds with integrated aptamer sequences, which enables direct construction of functional DNA origami structures. As we demonstrated, aptamers of various numbers and types were efficiently and stably integrated in user-defined positions of the scaffolds. Specifically, two different thrombin aptamer sequences were simultaneously inserted into the M13mp18 phage genome. The assembled functional DNA origami structures from this aptamer-integrated scaffold exhibited increased binding efficiency to thrombin and displayed more than 10-fold stronger resistance to exonuclease degradation than that produced using the traditional staple extension method. Additionally, a scaffold integrated with the platelet-derived growth factor aptamer was produced, and the assembled DNA origami structures showed significant inhibitory effect on breast cancer cells MDA-MB-231. This scalable method of creating design-specific scaffolds opens up a new way to construct more stable and functionally robust DNA origami structures and thus provides an important basis for their broader applications.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , DNA/química , Nanoestruturas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Conformação de Ácido Nucleico , Trombina/química
2.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361085

RESUMO

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure-activity relationship of sclerostin inhibitors for rational drug design.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Aptâmeros de Nucleotídeos/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Aptâmeros de Nucleotídeos/isolamento & purificação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteoporose/metabolismo , Osteoporose/patologia
3.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360665

RESUMO

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K+ or NH4+ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.


Assuntos
Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , DNA Catalítico/metabolismo , Fibrinogênio/metabolismo , Quadruplex G , Hemina/metabolismo , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Humanos , Modelos Moleculares
4.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445629

RESUMO

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Neoplasias/metabolismo , Receptores da Transferrina/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , Humanos , Ligantes , Neoplasias/patologia , Transcitose , Células Tumorais Cultivadas
5.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445583

RESUMO

Aptamers, single-stranded oligonucleotides that specifically bind a molecule with high affinity, are used as ligands in analytical and therapeutic applications. For the foodborne pathogen norovirus, multiple aptamers exist but have not been thoroughly characterized. Consequently, there is little research on aptamer-mediated assay development. This study characterized seven previously described norovirus aptamers for target affinity, structure, and potential use in extraction and detection assays. Norovirus-aptamer affinities were determined by filter retention assays using norovirus genotype (G) I.1, GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney virus-like particles. Of the seven aptamers characterized, equilibrium dissociation constants for GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney ranged from 71 ± 38 to 1777 ± 1021 nM. Four aptamers exhibited affinity to norovirus GII.4 strains; three aptamers additionally exhibited affinity toward GII.3 and GI.7. Aptamer affinity towards GI.1 was not observed. Aptamer structure analysis by circular dichroism (CD) spectroscopy showed that six aptamers exhibit B-DNA structure, and one aptamer displays parallel/antiparallel G-quadruplex hybrid structure. CD studies also showed that biotinylated aptamer structures were unchanged from non-biotinylated aptamers. Finally, norovirus aptamer assay feasibility was demonstrated in dot-blot and pull-down assays. This characterization of existing aptamers provides a knowledge base for future aptamer-based norovirus detection and extraction assay development and aptamer modification.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Proteínas do Capsídeo/metabolismo , Norovirus/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/isolamento & purificação , Bioensaio , Ligantes , Norovirus/química , Norovirus/genética
6.
Nat Commun ; 12(1): 4654, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341342

RESUMO

Ligand-oligonucleotide transduction provides the critical pathway to integrate non-nucleic acid molecules into nucleic acid circuits and nanomachines for a variety of strand-displacement related applications. Herein, a general platform is constructed to convert the signals of ligands into desired oligonucleotides through a precise kinetic control. In this design, the ligand-aptamer binding sequence with an engineered duplex stem is introduced between the toehold and displacement domains of the invading strand to regulate the strand-displacement reaction. Employing this platform, we achieve efficient transduction of both small molecules and proteins orthogonally, and more importantly, establish logical and cascading operations between different ligands for versatile transduction. Besides, this platform is capable of being directly coupled with the signal amplification systems to further enhance the transduction performance. This kinetically controlled platform presents unique features with designing simplicity and flexibility, expandable complexity and system compatibility, which may pave a broad road towards nucleic acid-based developments of sophisticated transduction networks.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Cinética , Ligantes , Modelos Genéticos , Modelos Moleculares , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Transdução de Sinais/genética , Espectrometria de Fluorescência/métodos
7.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206584

RESUMO

In this work, a simple and rapid method based on the lateral flow assay (LFA) has been developed for the detection of dual antibiotics. To achieve the quantitative assay and to reduce the non-specific adsorption, an internal system has been developed. A non-specific DNA was exploited as an internal standard and could be recognized by the DNA marker that was coated at the internal line. Two different kinds of aptamers were applied to recognize ampicillin (AMP) and kanamycin (KAM), and the distance between the detection line and conjugate pad was then optimized. Under the optimum conditions, the quantitative assays of AMP (R2 = 0.984) and KAM (R2 = 0.990) were achieved with dynamic ranges of 0.50 to 500.0 ng/L, and of 0.50 to 1000.0 ng/L, respectively. The LOQs of AMP and KAM were 0.06 ng/L and 0.015 ng/L, respectively. Finally, the proposed method has been successfully applied to analyze aquaculture water, tap water, and lake water, and hospital wastewater, indicating the established method could be used to monitor the environment.


Assuntos
Ampicilina/análise , Aptâmeros de Nucleotídeos/química , Canamicina/análise , Água/análise
8.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207373

RESUMO

A nucleic acid aptamer that specifically recognizes methicillin-resistant Staphylococcus aureus (MRSA) has been immobilized on magnetic nanoparticles to capture the target bacteria prior to mass spectrometry analysis. After the MRSA species were captured, they were further eluted from the nanoparticles and identified using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The combination of aptamer-based capture/enrichment and MS analysis of microorganisms took advantage of the selectivity of both techniques and should enhance the accuracy of MRSA identification. The capture and elution efficiencies for MRSA were optimized by examining factors such as incubation time, temperature, and elution solvents. The aptamer-modified magnetic nanoparticles showed a capture rate of more than 90% under the optimized condition, whereas the capture rates were less than 11% for non-target bacteria. The as-prepared nanoparticles exhibited only a 5% decrease in the capture rate and a 9% decrease in the elution rate after 10 successive cycles of utilization. Most importantly, the aptamer-modified nanoparticles revealed an excellent selectivity towards MRSA in bacterial mixtures. The capture of MRSA at a concentration of 102 CFU/mL remained at a good percentage of 82% even when the other two species were at 104 times higher concentration (106 CFU/mL). Further, the eluted MRSA bacteria were successfully identified using MALDI mass spectrometry.


Assuntos
Aptâmeros de Nucleotídeos/química , Nanopartículas de Magnetita/química , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus aureus Resistente à Meticilina/citologia , Técnica de Seleção de Aptâmeros/métodos
9.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206794

RESUMO

Herein, we have generated ssRNA aptamers to inhibit SARS-CoV-2 Mpro, a protease necessary for the SARS-CoV-2 coronavirus replication. Because there is no aptamer 3D structure currently available in the databanks for this protein, first, we modeled an ssRNA aptamer using an entropic fragment-based strategy. We refined the initial sequence and 3D structure by using two sequential approaches, consisting of an elitist genetic algorithm and an RNA inverse process. We identified three specific aptamers against SARS-CoV-2 Mpro, called MAptapro, MAptapro-IR1, and MAptapro-IR2, with similar 3D conformations and that fall in the dimerization region of the SARS-CoV-2 Mpro necessary for the enzymatic activity. Through the molecular dynamic simulation and binding free energy calculation, the interaction between the MAptapro-IR1 aptamer and the SARS-CoV-2 Mpro enzyme resulted in the strongest and the highest stable complex; therefore, the ssRNA MAptapro-IR1 aptamer was selected as the best potential candidate for the inhibition of SARS-CoV-2 Mpro and a perspective therapeutic drug for the COVID-19 disease.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , COVID-19/tratamento farmacológico , SARS-CoV-2/metabolismo , Proteínas da Matriz Viral/metabolismo , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , DNA de Cadeia Simples/química , Desenho de Fármacos , Entropia , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/química
10.
Nucleic Acids Res ; 49(13): 7267-7279, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34232998

RESUMO

We performed in vitro selection experiments to identify DNA aptamers for the S1 subunit of the SARS-CoV-2 spike protein (S1 protein). Using a pool of pre-structured random DNA sequences, we obtained over 100 candidate aptamers after 13 cycles of enrichment under progressively more stringent selection pressure. The top 10 sequences all exhibited strong binding to the S1 protein. Two aptamers, named MSA1 (Kd = 1.8 nM) and MSA5 (Kd = 2.7 nM), were assessed for binding to the heat-treated S1 protein, untreated S1 protein spiked into 50% human saliva and the trimeric spike protein of both the wildtype and the B.1.1.7 variant, demonstrating comparable affinities in all cases. MSA1 and MSA5 also recognized the pseudotyped lentivirus of SARS-CoV-2 with respective Kd values of 22.7 pM and 11.8 pM. Secondary structure prediction and sequence truncation experiments revealed that both MSA1 and MSA5 adopted a hairpin structure, which was the motif pre-designed into the original library. A colorimetric sandwich assay was developed using MSA1 as both the recognition element and detection element, which was capable of detecting the pseudotyped lentivirus in 50% saliva with a limit of detection of 400 fM, confirming the potential of these aptamers as diagnostic tools for COVID-19 detection.


Assuntos
Aptâmeros de Nucleotídeos , COVID-19/virologia , Biblioteca Gênica , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Sequência de Bases , COVID-19/diagnóstico , Colorimetria/métodos , Humanos , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros
11.
Nucleic Acids Res ; 49(13): 7280-7291, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34233000

RESUMO

Rational design of aptamers to incorporate unnatural nucleotides and special chemical moieties can expand their functional complexity and diversity. Spiegelmer (L-RNA aptamer) is a unique class of aptamer that is composed of unnatural L-RNA nucleotides, and so far there are limited L-RNA aptamer candidates and applications being reported. Moreover, the target binding properties of current L-RNA aptamers require significant improvement. Here, using L-Apt.4-1c as an example, we develop a simple and robust strategy to generate the first circular L-RNA aptamer, cycL-Apt.4-1c, quantitatively, demonstrate substantial enhancement in binding affinity and selectivity toward its target, and notably report novel applications of circular L-RNA aptamer in controlling RNA-protein interaction, and gene activity including telomerase activity and gene expression. Our approach and findings will be applicable to any L-RNA aptamers and open up a new avenue for diverse applications.


Assuntos
Aptâmeros de Nucleotídeos/química , RNA Circular/química , Química Click , Ciclização , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , RNA , Estabilidade de RNA , Telomerase/antagonistas & inibidores
12.
Artigo em Inglês | MEDLINE | ID: mdl-34219038

RESUMO

As an active glycoprotein with high nutritional value, lactoferrin is widely used in food and medical treatment. Therefore, it is very important to establish an accurate and efficient detection method for lactoferrin. At present, the detection of lactoferrin in milk faces many challenges, such as low separation degree and poor parallelism. To address this issue, we developed an aptamer affinity column (AAC) for purification and enrichment of lactoferrin in milk. The column was prepared by covalent conjugation of an amino-modified aptamer with NHS-activated Sepharose. The washing buffer type (0.01 mol/L phosphate buffer) and volume (10 mL) and the sodium chlorideconcentration (1 mol/L) in the elution buffer were optimized for the AAC method. The performance of the AAC was then evaluated in terms of the column capacity, specificity, stability, and reusability. The column capacity was 500 ± 13.7 µg and the column could be reused up to ten times with a large loss in performance. The AAC method combined with high-performance liquid chromatography gave excellent linearity over a wide range, good sensitivity with a limit of detection of 3 µg/mL, and acceptable recoveries for different concentrations of lactoferrin spiked in real raw milk samples from cattle. Finally, the AAC was successfully applied to analyze lactoferrin in milk. This method could be applied to routine analysis of samples for lactoferrin in testing laboratories and dairy factories.


Assuntos
Cromatografia de Afinidade/métodos , Lactoferrina , Leite/química , Animais , Aptâmeros de Nucleotídeos/química , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Lactoferrina/análise , Lactoferrina/química , Lactoferrina/isolamento & purificação , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
13.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208896

RESUMO

In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.


Assuntos
Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Neoplasias Colorretais/genética , Neoplasias Pulmonares/genética , Timidina/genética , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G , Células HCT116 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Espectroscopia de Ressonância Magnética
14.
Se Pu ; 39(7): 721-729, 2021 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-34227370

RESUMO

8-Oxoguanine DNA glycosylase (OGG1) is an important enzyme that plays a key role in oxidative DNA damage repair. OGG1 can specifically recognize and excise 8-oxoG (a product of oxidative damage found in double-stranded DNA) through base excision repair (BER). OGG1 is expressed in normal tissues, and in most tumor tissues. Oxidative cellular damage can produce an inflammatory reaction, alleviating some measure of constitutive OGG1 inhibition. OGG1 inhibition in cancer cells shows some promise as a new method of cancer treatment. Most current OGG1 research focuses on regulating OGG1 with targeted small molecules. To date, no aptamer screen for OGG1 has been reported. Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target with high affinity and specificity in vitro, that can be identified by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used as chemical ligands to regulate intermolecular interactions. In this study, a screen for aptamers with OGG1 affinity was performed for the first time. Capillary electrophoresis (CE) is a microanalytical technique that offers speed and high separation efficiency. In this work, two screening methods based on CE-SELEX technology were established: a one-round pressure controllable selection, and a multi-round selection. The most important criterion for successful one-round pressure controllable selection is to select a competitive target with a different CE migration time than that of the target of interest. We mixed OGG1 with a competitive target and a nucleic acid library for CE analysis. Two proteins competitively bind sequences in the library, forming independent complexes. The concentration of the competitive target is continuously increased until complexes with the target stop decreasing, indicating that the target and the ssDNA library have formed a stable complex. Complexes were collected for PCR amplification, purification, and high-throughput sequencing to obtain high affinity aptamers. This method greatly improves screening efficiency, and reduces non-specific binding to the target, which is helpful for obtaining aptamers with high affinity and specificity. One-round pressure controllable selection for high affinity OGG1 selective aptamers was performed using single strand binding protein (SSB) to competitively and tightly bind nucleic acids in the library. The competitive screening pressure was increased by increasing the SSB concentration to eliminate sequences with low affinity for OGG1 from the random oligonucleotide library. Nucleic acid sequences with high OGG1 affinity were obtainable in one step, and OGG1-ssDNA complexes were collected by creating a timed program on Beckman P/ACE MDQ capillary electrophoresis. Collection occurred from 2.2 to 2.8 min. Under identical incubation and electrophoresis conditions, multiple round selections were conducted by injecting samples of co-incubated nucleic acid library and target into the capillary. After separation under a high-voltage electric field, nucleic acid target complexes were collected, amplified by PCR, purified, and used as an enriched secondary library in the next round of screening. High affinity aptamers were generally obtained within three rounds. Comparing results of the two screening methods, the three candidate aptamer sequences found with the highest frequency were consistent, and displayed KD values ranging from 1.71 to 2.64 µmol/L. Molecular docking analysis suggests that Apt 1 may bind to the OGG1 active pocket, which functions to repair oxidative damage. Comparison of the two screening methods indicates that one-round pressure controllable selection is more rapid and efficient, providing guidance for the design of other protein aptamer screening methods. The obtained aptamer is expected to be function effectively as an OGG1-mediated DNA repair inhibitor.


Assuntos
Aptâmeros de Nucleotídeos , DNA Glicosilases , Aptâmeros de Nucleotídeos/química , DNA Glicosilases/química , DNA de Cadeia Simples , Eletroforese Capilar , Guanina/análogos & derivados , Simulação de Acoplamento Molecular , Técnica de Seleção de Aptâmeros
15.
Angew Chem Int Ed Engl ; 60(39): 21211-21215, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34328683

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×105  copies mL-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.


Assuntos
Aptâmeros de Nucleotídeos/química , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/análise , COVID-19/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Modelos Moleculares , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
17.
Nat Commun ; 12(1): 3549, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112799

RESUMO

Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Guanina/química , RNA/química , Sítios de Ligação , Cristalografia , Fluorescência , Ligação de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutagênese , Mutação , Prótons , Espectrometria de Fluorescência
18.
Nat Commun ; 12(1): 3877, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162884

RESUMO

Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand's methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.


Assuntos
Conformação de Ácido Nucleico , Nucleosídeo Q/química , RNA/química , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Cinética , Metilação , Estrutura Molecular , Nucleosídeo Q/metabolismo , RNA/genética , RNA/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo
19.
Nucleic Acids Res ; 49(11): 6069-6081, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34095949

RESUMO

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Luminol/metabolismo , Mioglobina/metabolismo , Peroxidase/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Simulação por Computador , Heme/metabolismo , Luminescência , Luminol/química , Ressonância Magnética Nuclear Biomolecular , Técnica de Seleção de Aptâmeros , Especificidade por Substrato
20.
Food Chem ; 362: 130261, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111691

RESUMO

In this study, a novel surface enhanced Raman spectroscopy (SERS) sensor was developed for the ultrasensitive determination of kanamycin in foods. The sensor used two distinct signal amplification strategies, namely the surface plasmon resonance of gold nanorods and a Zn-doped carbon quantum dots catalytic cascade oxidation-reduction reaction switch controlled by a nucleic acid aptamer. Under optimized experimental conditions, the SERS sensor demonstrated a linear range of 10-12 to 10-5 g mL-1 for the detection of kanamycin, with a limit of detection of 3.03 × 10-13 g mL-1. Experiments with antibiotics structurally similar to kanamycin and interferrants revealed that the sensor had excellent selectivity. Milkpowder and honey samples spiked with kanamycin were assayed, with recoveries ranging from 84.1% to 107.2% and a relative standard deviation of 0.74% to 2.81% being obtained. Quantification of kanamycin in milk samples revealed no significant difference between the results obtained with the sensor and by HPLC.


Assuntos
Aptâmeros de Nucleotídeos/química , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Canamicina/análise , Nanotubos/química , Pontos Quânticos/química , Zinco/química , Antibacterianos/análise , Técnicas Biossensoriais/instrumentação , Carbono/química , Catálise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície
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