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1.
Nat Commun ; 11(1): 4384, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873796

RESUMO

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , DNA/química , Nanoestruturas/química , Imagem Individual de Molécula/instrumentação , Biomarcadores/análise , Proteína C-Reativa/análise , Desenho de Equipamento , Humanos , Limite de Detecção , Nanotecnologia/métodos
2.
Chem Commun (Camb) ; 56(70): 10235-10238, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32756614

RESUMO

Here, we report for the first time DNA aptamers targeted toward the COVID-19 nucleocapsid protein (Np). Np is one of the most abundant structural proteins and it serves as a diagnostic marker for the accurate and sensitive detection of COVID-19. After five rounds of selection, we obtained four DNA sequences with an affinity below 5 nM. The best one displayed a superb binding performance toward Np with a Kd value of 0.49 nM. Interestingly, we found that the four pairs of aptamers could bind to Np successively, suggesting a sandwich-type interaction. Using these sandwiched aptamers in ELISA and colloidal gold immunochromatographic strips, we were able to detect Np at the tens of pM level. The results demonstrate that aptamers are powerful molecular tools for virus detection, diagnosis, and antiviral therapy.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Betacoronavirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Ouro/química , Humanos , Cinética , Limite de Detecção , Nanopartículas Metálicas/química , Proteínas do Nucleocapsídeo/química , Pandemias , Pneumonia Viral/diagnóstico , Técnica de Seleção de Aptâmeros
3.
PLoS One ; 15(8): e0237253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790805

RESUMO

Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing. After three rounds of selections, a highly enriched ssDNA pool was sequenced, and the results were analyzed using FASTAptamer Toolkit. Sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. Top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (ELAA) for rapid and sensitive detection of NDV in farm samples. The selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. Following optimization of the sandwich ELAA method, the results demonstrated that both selected aptamers Apt_NDV01 and Apt_NDV03 with dissociation constant values of 31 nM and 78.1 nM, respectively, showed the highest specificity and affinity for NDV detection. The ELAA results were verified by quantitative real-time PCR, demonstrating strong concordance, and showing outstanding accuracy for detection of NDV in field sample. In summary, combination of SELEX with high-throughput DNA sequencing allowed rapid screening and selection of aptamers. The selected aptamers allowed recognition of NDV with high affinities. This is the first report that uses a validated sandwich ELAA for rapid and specific detection of NDV in poultry samples.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/isolamento & purificação , Animais , Técnicas Biossensoriais , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Newcastle/virologia , Aves Domésticas/virologia , Técnica de Seleção de Aptâmeros
4.
Nat Commun ; 11(1): 3847, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737299

RESUMO

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.


Assuntos
Aptâmeros de Nucleotídeos/genética , Arabidopsis/genética , Brassica/genética , Engenharia Genética/métodos , RNA Mensageiro/genética , Tabaco/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Arabidopsis/metabolismo , Compostos de Benzil/química , Brassica/metabolismo , Fluorescência , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Genes Reporter , Imidazolinas/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/metabolismo , Tabaco/metabolismo , Transformação Genética
5.
Biosensors (Basel) ; 10(9)2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847008

RESUMO

Cytokines are a family of proteins which play a major role in the regulation of the immune system and the development of several diseases, from rheumatoid arthritis to cancer and, more recently, COVID-19. Therefore, many efforts are currently being developed to improve therapy and diagnosis, as well as to produce inhibitory drugs and biosensors for a rapid, minimally invasive, and effective detection. In this regard, even more efficient cytokine receptors are under investigation. In this paper we analyze a set of IL-6 cytokine receptors, investigating their topological features by means of a theoretical approach. Our results suggest a topological indicator that may help in the identification of those receptors having the highest complementarity with the protein, a feature expected to ensure a stable binding. Furthermore, we propose and discuss the use of these receptors in an idealized experimental setup.


Assuntos
Técnicas Biossensoriais/métodos , Interleucina-6/análise , Receptores de Interleucina-6/análise , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Aptâmeros de Nucleotídeos/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/imunologia , Limite de Detecção , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Receptores de Interleucina-6/imunologia
6.
Int J Nanomedicine ; 15: 4237-4256, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606675

RESUMO

With the passage of time and more advanced societies, there is a greater emergence and incidence of disease and necessity for improved treatments. In this respect, nowadays, aptamers, with their better efficiency at diagnosing and treating diseases than antibodies, are at the center of attention. Here, in this review, we first investigate aptamer function in various fields (such as the detection and remedy of pathogens, modification of nanoparticles, antibiotic delivery and gene delivery). Then, we present aptamer-conjugated nanocomplexes as the main and efficient factor in gene delivery. Finally, we focus on the targeted co-delivery of genes and drugs by nanocomplexes, as a new exciting approach for cancer treatment in the decades ahead to meet our growing societal needs.


Assuntos
Antibacterianos/farmacologia , Aptâmeros de Nucleotídeos/química , Técnicas de Transferência de Genes , Nanopartículas/química , Sistemas de Liberação de Medicamentos , Humanos , Nanopartículas/ultraestrutura , Polietilenoimina/química
7.
PLoS One ; 15(7): e0235328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628701

RESUMO

OBJECTIVE: Current urinary tract infection (UTI) diagnostic strategies that rely on leukocyte esterase have limited accuracy. We performed an aptamer-based proteomics pilot study to identify urine protein levels that could differentiate a culture proven UTI from culture negative samples, regardless of pyuria status. METHODS: We analyzed urine from 16 children with UTIs, 8 children with culture negative pyuria and 8 children with negative urine culture and no pyuria. The urine levels of 1,310 proteins were quantified using the Somascan™ platform and normalized to urine creatinine. Machine learning with support vector machine (SVM)-based feature selection was performed to determine the combination of urine biomarkers that optimized diagnostic accuracy. RESULTS: Eight candidate urine protein biomarkers met filtering criteria. B-cell lymphoma protein, C-X-C motif chemokine 6, C-X-C motif chemokine 13, cathepsin S, heat shock 70kDA protein 1A, mitogen activated protein kinase, protein E7 HPV18 and transgelin. AUCs ranged from 0.91 to 0.95. The best prediction was achieved by the SVMs with radial basis function kernel. CONCLUSIONS: Biomarkers panel can be identified by the emerging technologies of aptamer-based proteomics and machine learning that offer the potential to increase UTI diagnostic accuracy, thereby limiting unneeded antibiotics.


Assuntos
Aptâmeros de Nucleotídeos/química , Proteômica/métodos , Máquina de Vetores de Suporte , Urinálise/métodos , Infecções Urinárias/diagnóstico , Adolescente , Biomarcadores/urina , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Projetos Piloto , Estudos Prospectivos , Infecções Urinárias/urina
8.
Nucleic Acids Res ; 48(15): e90, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32609809

RESUMO

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição de Choque Térmico/química , Substâncias Macromoleculares/isolamento & purificação , Espectrometria de Massas , Aptâmeros de Nucleotídeos/genética , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/genética , Histonas/química , Humanos , Imunoprecipitação , Substâncias Macromoleculares/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional
9.
Proc Natl Acad Sci U S A ; 117(29): 16790-16798, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631977

RESUMO

Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.


Assuntos
Aptâmeros de Nucleotídeos/química , L-Lactato Desidrogenase/química , Malária/diagnóstico , Proteínas de Protozoários/química , Biomarcadores/sangue , Biomarcadores/química , Humanos , Ligação de Hidrogênio , L-Lactato Desidrogenase/sangue , Malária/sangue , Técnicas de Diagnóstico Molecular/métodos , Simulação de Dinâmica Molecular , Plasmodium vivax/enzimologia , Ligação Proteica
10.
J Vis Exp ; (160)2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32658190

RESUMO

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.


Assuntos
Aptâmeros de Nucleotídeos/química , Doxorrubicina/química , Portadores de Fármacos/química , Portadores de Fármacos/síntese química , Ouro/química , Nanopartículas Metálicas/química , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Células A549 , Técnicas de Química Sintética , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Humanos , Oligodesoxirribonucleotídeos/química , Polietilenoimina/química
11.
BMC Bioinformatics ; 21(1): 236, 2020 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-32517696

RESUMO

BACKGROUND: The interactions between proteins and aptamers are prevalent in organisms and play an important role in various life activities. Thanks to the rapid accumulation of protein-aptamer interaction data, it is necessary and feasible to construct an accurate and effective computational model to predict aptamers binding to certain interested proteins and protein-aptamer interactions, which is beneficial for understanding mechanisms of protein-aptamer interactions and improving aptamer-based therapies. RESULTS: In this study, a novel web server named PPAI is developed to predict aptamers and protein-aptamer interactions with key sequence features of proteins/aptamers and a machine learning framework integrated adaboost and random forest. A new method for extracting several key sequence features of both proteins and aptamers is presented, where the features for proteins are extracted from amino acid composition, pseudo-amino acid composition, grouped amino acid composition, C/T/D composition and sequence-order-coupling number, while the features for aptamers are extracted from nucleotide composition, pseudo-nucleotide composition (PseKNC) and normalized Moreau-Broto autocorrelation coefficient. On the basis of these feature sets and balanced the samples with SMOTE algorithm, we validate the performance of PPAI by the independent test set. The results demonstrate that the Area Under Curve (AUC) is 0.907 for prediction of aptamer, while the AUC reaches 0.871 for prediction of protein-aptamer interactions. CONCLUSION: These results indicate that PPAI can query aptamers and proteins, predict aptamers and predict protein-aptamer interactions in batch mode precisely and efficiently, which would be a novel bioinformatics tool for the research of protein-aptamer interactions. PPAI web-server is freely available at http://39.96.85.9/PPAI.


Assuntos
Aminoácidos/metabolismo , Aptâmeros de Nucleotídeos/química , Biologia Computacional/métodos , Proteínas/química , Humanos
12.
BMC Bioinformatics ; 21(1): 263, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32580745

RESUMO

BACKGROUND: The combination of systematic evolution of ligands by exponential enrichment (SELEX) and deep sequencing is termed high-throughput (HT)-SELEX, which enables searching aptamer candidates from a massive amount of oligonucleotide sequences. A clustering method is an important procedure to identify sequence groups including aptamer candidates for evaluation with experimental analysis. In general, aptamer includes a specific target binding region, which is necessary for binding to the target molecules. The length of the target binding region varies depending on the target molecules and/or binding styles. Currently available clustering methods for HT-SELEX only estimate clusters based on the similarity of full-length sequences or limited length of motifs as target binding regions. Hence, a clustering method considering the target binding region with different lengths is required. Moreover, to handle such huge data and to save sequencing cost, a clustering method with fast calculation from a single round of HT-SELEX data, not multiple rounds, is also preferred. RESULTS: We developed fast string-based clustering (FSBC) for HT-SELEX data. FSBC was designed to estimate clusters by searching various lengths of over-represented strings as target binding regions. FSBC was also designed for fast calculation with search space reduction from a single round, typically the final round, of HT-SELEX data considering imbalanced nucleobases of the aptamer selection process. The calculation time and clustering accuracy of FSBC were compared with those of four conventional clustering methods, FASTAptamer, AptaCluster, APTANI, and AptaTRACE, using HT-SELEX data (>15 million oligonucleotide sequences). FSBC, AptaCluster, and AptaTRACE could complete the clustering for all sequence data, and FSBC and AptaTRACE performed higher clustering accuracy. FSBC showed the highest clustering accuracy and had the second fastest calculation speed among all methods compared. CONCLUSION: FSBC is applicable to a large HT-SELEX dataset, which can facilitate the accurate identification of groups including aptamer candidates. AVAILABILITY OF DATA AND MATERIALS: FSBC is available at http://www.aoki.ecei.tohoku.ac.jp/fsbc/.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Análise por Conglomerados , Software
13.
RNA ; 26(9): 1283-1290, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32482894

RESUMO

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.


Assuntos
Aptâmeros de Nucleotídeos/química , Corantes Fluorescentes/química , Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Replicação de Sequência Autossustentável/métodos , Sistema Livre de Células , Fluorescência , Humanos , Regiões Promotoras Genéticas/genética , Sensibilidade e Especificidade
14.
Nucleic Acids Res ; 48(14): e82, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32537639

RESUMO

Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/isolamento & purificação , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Simulação por Computador , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Curva ROC
15.
Nucleic Acids Res ; 48(13): 7569-7583, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32544228

RESUMO

Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure. It suggests that cobalamin selectivity might ultimately be viewed as existing on a spectrum of affinity for each derivative rather than as belonging to distinct types based on ligand specificities. Our work provides an interesting and notable example of functional coupling of ligand-sensing and adaptive folding by a structured RNA molecule.


Assuntos
Aptâmeros de Nucleotídeos/química , Cobamidas/química , Dobramento de RNA , Riboswitch , Vitamina B 12/química , Bacillus subtilis
16.
Food Chem ; 330: 127230, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526651

RESUMO

Chitooligosaccharides are oligosaccharides with many biological activities that can be used in food production for sweeteners, preservatives and humectants, among other products. Chitin, a long-chain polymer of N-acetylglucosamine and a derivative of glucose, can be hydrolyzed by applying chitinase to break down glycosidic bonds to form chitooligosaccharides. Chitinases arising from heterologous gene expression are usually linked to a 6 × His-tag to facilitate easy purification. Heterologously expressed chitinase linked to a 6 × His-tag is a transgenic element, but enzyme activity tests cannot be used to distinguish transgenic elements from natural elements. In this study, we established a rapid and easy method to detect His-tag-containing chitinase using gold nanoparticles (AuNPs) and ssDNA aptamers. Using this method, His-tag-containing chitinase could be detected at concentrations as low as 0.136 nM within 5 min. Color changes of AuNPs showed a positive correlation with His-tag-containing chitinase concentrations.


Assuntos
Aptâmeros de Nucleotídeos/química , Quitinases/metabolismo , DNA de Cadeia Simples/química , Ouro/química , Nanopartículas Metálicas/química , Quitina/análogos & derivados , Quitina/metabolismo , Cor , Hidrólise , Fatores de Tempo
17.
Nucleic Acids Res ; 48(12): 6970-6979, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479610

RESUMO

Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.


Assuntos
Aptâmeros de Nucleotídeos/genética , Conformação de Ácido Nucleico , Ribonucleotídeos/genética , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Firmicutes/genética , Firmicutes/ultraestrutura , Ligantes , Purinas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Ribonucleotídeos/química
18.
Nucleic Acids Res ; 48(12): 6431-6444, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442276

RESUMO

While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.


Assuntos
Aptâmeros de Nucleotídeos/química , Trombina/química , Fator A de Crescimento do Endotélio Vascular/química , Técnicas Biossensoriais/métodos , Humanos , Ligação Proteica , Trombina/análise , Fator A de Crescimento do Endotélio Vascular/análise
19.
Nucleic Acids Res ; 48(12): 6471-6480, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442296

RESUMO

Despite their great success in recognizing small molecules in vitro, nucleic acid aptamers are rarely used in clinical settings. This is partially due to the lack of structure-based mechanistic information. In this work, atomistic molecular dynamics simulations are used to study the static and dynamic supramolecular structures relevant to the process of the wild-type (wt) nucleic acid aptamer recognition and binding of ATP. The effects brought about by mutation of key residues in the recognition site are also explored. The simulations reveal that the aptamer displays a high degree of rigidity and is structurally very little affected by the binding of ATP. Interaction energy decomposition shows that dispersion forces from π-stacking between ATP and the G6 and A23 nucleobases in the aptamer binding site plays a more important role in stabilizing the supramolecular complex, compared to hydrogen-bond interaction between ATP and G22. Moreover, metadynamics simulations show that during the association process, water molecules act as essential bridges connecting ATP with G22, which favors the dynamic stability of the complex. The calculations carried out on three mutated aptamer structures confirm the crucial role of the hydrogen bonds and π-stacking interactions for the binding affinity of the ATP nucleic acid aptamer.


Assuntos
Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Simulação de Dinâmica Molecular , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , Ligação de Hidrogênio , Mutação
20.
RNA ; 26(8): 982-995, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32371455

RESUMO

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.


Assuntos
Aptâmeros de Nucleotídeos/química , RNA Catalítico/química , RNA/química , Sequência de Bases , Ligantes , Conformação de Ácido Nucleico , Riboswitch/genética
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