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1.
Carbohydr Polym ; 296: 119905, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36088018

RESUMO

A nature-inspired strategy is developed to build dual-network hydrogels made up of rigid graphene oxide-functionalized nanocellulose (GO@NC) network and flexible poly[acrylamide-co-(acrylic acid)] (poly(AAm-co-AAc)) network. A pre-stretching method is used to form a muscle-shape anisotropic architecture. The penetration of poly(AAm-co-AAc) flexible network relieves the stiffness of NC network, thus improving the average elongation at break from 86.2 % to 748.0 %. Compared with the poly(AAm-co-AAc), the average rupture tensile strength rises remarkably by 228.6 %. The dual-crosslinked strategy endows the GO@NC-poly(AAm-co-AAc) hydrogels with a fast, stable and repeatable self-healing ability, which can achieve 85.0 % of healing efficiency after only 600 s of self-healing and maintain 76.2 % of initial strength after 10 cycles of breaking-self-healing. The superb self-healing ability is similar to the muscle function. For potential applications, the hydrogels can achieve real-time, stable, and long-term sensing as smart wearable strain sensors (high gauge factor: 5.13), and can effectively purify Sudan IV wastewater as green recyclable adsorbents.


Assuntos
Grafite , Hidrogéis , Acrilamidas , Poli A , Resistência à Tração
2.
Sci Rep ; 12(1): 15180, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36071149

RESUMO

In this work, we looked at how to make fluorescence hybrid poly(amidoamine) dendrimer (PAMAM) dendrimers using calcozine red 6G and coumarin end groups. After synthesis of ethylenediamine (EDA)-cored 4th generation PAMAM dendrimer (G4.0), surface functional groups is reacted with calcozine red 6G (Rh6G) and 7-methacryloyloxy-4-methylcoumarin. Fourier transform infrared spectroscopy, proton nuclear magnetic resonance (1H NMR), and X-ray diffraction are used to characterize the structure of synthesized fluorescent hybrid dendrimers. Optical properties are demonstrated using a fluorescence spectrophotometer, and UV-Vis-NIR reflectance spectra. According to UV-Vis-NIR reflectance spectra, hybrid dendrimers were transparent in the NIR range. Moreover, quantum yield (Φs) of hybrid dendrimers was calculated in dimethylformamide (DMF), ethanol, dimethyl sulfoxide (DMSO), and distilled water (H2O). Dendrimers in which Rh6G was utilized to modification showed the maximum quantum yield in ethanol due to great interaction of structure with ethanol and the arrangement of ring-opened amide shape of calcozine red 6G.


Assuntos
Dendrímeros , Dendrímeros/química , Etanol , Fluorescência , Poli A , Poliaminas/química
3.
J Phys Chem B ; 126(36): 6948-6954, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36027577

RESUMO

Hydrogen bonding between the DNA nucleobases and small organic molecules, such as melamine, is a new strategy for the design of novel DNA materials. Poly(thymidine) DNA and melamine self-assemble into a duplex structure containing two antiparallel DNA strands hydrogen bonded to central melamine units. In this Article, molecular dynamics simulations rationalize the observed antiparallel duplex structure. Alternative duplex and triplex structures with parallel and antiparallel strand orientations are shown to be unstable because of the increase in unfavorable interactions between the DNA backbones.


Assuntos
DNA , Triazinas , DNA/química , Conformação de Ácido Nucleico , Poli A , Timidina
4.
Mol Cell Biol ; 42(9): e0024422, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35972270

RESUMO

The 3' ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3' untranslated regions (3' UTRs) but rarely within coding regions. An individual gene can yield many 3'-mRNA isoforms with distinct half-lives. We dissect the relative contributions of protein-coding sequences (open reading frames [ORFs]) and 3' UTRs to polyadenylation profiles in yeast. ORF-deleted derivatives often display strongly decreased mRNA levels, indicating that ORFs contribute to overall mRNA stability. Poly(A) profiles, and hence relative isoform half-lives, of most (9 of 10) ORF-deleted derivatives are very similar to their wild-type counterparts. Similarly, in-frame insertion of a large protein-coding fragment between the ORF and 3' UTR has minimal effect on the poly(A) profile in all 15 cases tested. Last, reciprocal ORF/3'-UTR chimeric genes indicate that the poly(A) profile is determined by the 3' UTR. Thus, 3' UTRs are self-contained modular entities sufficient to determine poly(A) profiles and relative 3'-isoform half-lives. In the one atypical instance, ORF deletion causes an upstream shift of poly(A) sites, likely because juxtaposition of an unusually high AT-rich stretch directs polyadenylation closely downstream. This suggests that long AT-rich stretches, which are not encountered until after coding regions, are important for restricting polyadenylation to 3' UTRs.


Assuntos
Poliadenilação , Isoformas de RNA , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas , Poli A/genética , Isoformas de Proteínas/genética , Isoformas de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Phys Chem Chem Phys ; 24(32): 19552-19563, 2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-35938929

RESUMO

DNA mediated directed self assembly of gold nanoparticles (AuNPs) has garnered immense interest due to its ability to precisely control supramolecular assemblies. Most experimental works have relied on utilizing the complementary interactions between the DNA strands to drive the self assembly of AuNPs grafted with DNA strands. In the present work, we have leveraged DNA-peptide interactions to tune the self assembly and stimuli responsive behavior of AuNPs grafted with single stranded DNA (ssDNA) and poly-L-lysine (PLL) chains. Our findings show that the electrostatic interactions between the negatively charged ssDNA grafts and positively charged PLL grafts, drive the self assembly of AuNPs of different sizes into 3D nanostructures. The transmission electron micrographs confirm that the smaller AuNPs grafted with PLL chains form a corona around the large AuNPs grafted with ssDNA like the petals around a flowery core to drive aggregation of large AuNPs. When the grafting of ssDNA and PLL on the different sized AuNPs is swapped, aggregates of large AuNPs mediated by the ssDNA grafts on the smaller AuNPs were observed. The presence of excess ssDNA/PLL chains in solutions affected both the morphology and the mechanism of aggregate formation. Coarse-grain molecular dynamics simulations qualitatively match the experimental findings and provided a scientific rationale to the above findings highlighting the role of chain entropy, molecular connectivity, and charge correlations on the self assembly of AuNPs.


Assuntos
Ouro , Nanopartículas Metálicas , DNA/química , DNA de Cadeia Simples , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/química , Poli A
6.
Langmuir ; 38(32): 9833-9843, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35916504

RESUMO

In this study, we utilized selectively modified, biodegradable polymer-based polyplexes to deliver custom, exogenous miR-148b mimics to induce apoptosis in human lung cancer (A549) cells. The gene regulatory effects of the payload miRNA mimics (miR-148b-3p) were first evaluated through bioinformatic analyses to uncover specific gene targets involved in critical carcinogenic pathways. Hyperbranched poly(ß amino ester) polyplexes (hPBAE) loaded with custom miR-148b mimics were then developed for targeted therapy. When evaluated in vitro, these hPBAE-based polyplexes sustained high intracellular uptake, low cytotoxicity, and efficient escape from endosomes to deliver functionally intact miRNA mimics to the cytosol. High-resolution confocal microscopy revealed successful intracellular uptake, cell viability was assessed through qualitative fluorescence microscopy and fluorescence-based DNA quantification, and successful cytosolic delivery of intact miRNA mimics was evaluated using real-time polymerase chain reaction (RT-PCR) to demonstrate target gene knockdown. The hPBAE-miRNA mimic polyplexes were shown to induce apoptosis among A549 cells through direct modulation of intracellular protein expression, targeting multiple potential carcinogenic pathways at the gene level. These results indicated that spatially controlled miR-148b mimic delivery can promote efficient cancer cell death in vitro and may lead to an enhanced therapeutic design for in vivo application.


Assuntos
Ésteres , MicroRNAs , Células A549 , Apoptose , Proliferação de Células , Humanos , MicroRNAs/genética , Poli A , Polímeros
7.
Anal Chem ; 94(36): 12342-12351, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36018770

RESUMO

The occurrence of diseases displayed transcriptome alteration, including both coding and non-coding transcripts. The third-generation sequencing (TGS) technologies allow for intensive and comprehensive research of the transcriptome. However, the present standard TGS RNA sequencing method is unable to detect many of the non-polyadenylated [non-poly(A)] RNAs. To obtain more complete transcriptome information, we presented a new comprehensive sequencing approach by performing conventional poly(A) RNA-sequencing combined with the sequencing of non-poly(A) RNA fraction which was tailed by poly(U) on HepG2 and HL-7702 cell lines, enabling the detection of multiple categories of non-poly(A) RNAs excluded by the existing standard approach. Moreover, the length distribution of the full-splice match transcripts was longer than that assembled by short-reads, which contributed to characterizing alternative splicing events and provided a comprehensive portrait of transcriptional complexity. Besides the detection of genes with differential expression patterns in the poly(A) library between HepG2 and HL-7702, we also found a cancer-related non-coding gene in the poly(U) data, that is, growth arrest special 5 (GAS5). Collectively, our results suggested that the novel method effectively captured both poly(A) and non-poly(A) transcripts in the tested cell lines and allowed a deeper exploration of the transcriptome.


Assuntos
Sequenciamento por Nanoporos , RNA , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Poli A/genética , RNA/genética , RNA Mensageiro/genética , RNA-Seq , Análise de Sequência de RNA , Transcriptoma
8.
Plant Sci ; 324: 111430, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36007628

RESUMO

Recent years have seen an explosion of interest in the subject of alternative polyadenylation in plants. Connections between the polyadenylation complex and numerous developmental and stress responses are well-established. However, those that link stimuli with the functioning of the polyadenylation complex are less well understood. To this end, it is imperative to clearly delineate the roles of the polyadenylation complex in both plant growth AND alternative polyadenylation. It is also necessary to understand the ways by which other molecular processes may contribute to alternative polyadenylation. This review discusses these issues, with a focus on instances that reveal mechanisms by which mRNA polyadenylation may be regulated. Insights from from characterizations of mutants affected in the polyadenylation complex are discussed, as are the limitations of such characterizations when it comes to teasing out cause and effect. These limitations encourage explorations to other processes that are beyond the core polyadenylation complex. Two such processes that sculpt the plant transcriptome - transcription termination and the epigenetic control of transposon activity - also contribute to regulated poly(A) site choice. These subjects define "the right places" - molecular mechanisms that contribute to the wide-ranging control of gene expression via mRNA polyadenylation.


Assuntos
Poli A , Poliadenilação , Humanos , Plantas/genética , Plantas/metabolismo , Poli A/genética , Poli A/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
9.
Int J Biol Macromol ; 219: 579-586, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-35952809

RESUMO

RNA triplexes have a variety of potential applications in molecular biology, diagnostics and therapeutics, while low stabilization of the third strand hinders their practical utilities under physiological conditions. In this regard, achieving the third-strand stabilization by binding small molecules is a promising strategy. Chirality is one of the basic properties of nature. To clarify achirality and chirality effects on the binding and stabilizing effects of RNA triplexes by small molecules, we report for the first time the RNA interactions of an racemic ruthenium(II) polypyridyl complex [Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) and its two enantiomers Δ/Λ-[Ru(bpy)2(11-CN-dppz)]2+ (Δ/Λ-Ru1) with an RNA triplex poly(U-A*U) (where "-" represents Watson-Crick base pairing, and "*" denotes Hoogsteen base pairing, respectively) in this work. Research shows that although rac-Ru1 and its two enantiomers Δ/Λ-Ru1 bind to the RNA triplex through the same mode of intercalation, the binding affinity for enantiomer Δ-Ru1 is much higher than that for rac-Ru1 and enantiomer Λ-Ru1. However, compared to enantiomer Λ-Ru1, the binding affinity for rac-Ru1 does not show much of an advantage, which is slightly greater than that for the former. Thermal denaturation measurements reveal both rac-Ru1 and Δ-Ru1 to have a preference for stabilizing the third strand rather than the template duplex of the RNA triplex, while Λ-Ru1 stabilizes the RNA triplex without significant selectivity. Besides, the third-strand stabilizing effects by rac-Ru1 and Δ-Ru1 are not markedly different from each other, but more marked than that by Λ-Ru1. This work shows that the binding properties of the racemic Ru(II) polypyridyl complex with the RNA triplex are not simply an average of its two enantiomers, indicating potentially complicated binding events.


Assuntos
Rutênio , Conformação de Ácido Nucleico , Poli A/química , Poli U/química , RNA/química , Rutênio/química
10.
Mol Cell ; 82(17): 3135-3150.e9, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35914531

RESUMO

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.


Assuntos
Poli A , Poliadenilação , Regiões 3' não Traduzidas , Humanos , Poli A/metabolismo , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Zinco/metabolismo
11.
J Phys Chem Lett ; 13(29): 6701-6710, 2022 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-35848986

RESUMO

Nature has beautifully assembled its light harvesting pigments within protein scaffolds, which ensures a very high energy transfer. Designing a highly efficient artificial bioinspired light harvesting system (LHS) thus requires the nanoscale spatial orientation and electronic control of the associated chromophores. Although DNA has been used as a scaffold to organize chromophores, proteins or polypeptides, however, are very rarely explored. Here, we have developed a highly efficient, artificial, bioinspired LHS using polypeptide (poly-d-lysine, PDL) nanostructures making use of their ß-sheet structure in an aqueous alkaline medium. The chromophores used herein are compatible for an energy transfer process and are nonfluorescent in an aqueous medium but exhibit high fluorescence intensity when bound to the nanostructure of PDL. The close proximity of the chromophores results in an energy transfer efficiency of ∼92% besides generating white light emission at a particular molar ratio between the chromophores.


Assuntos
Complexos de Proteínas Captadores de Luz , Nanoestruturas , Transferência de Energia , Luz , Complexos de Proteínas Captadores de Luz/química , Lisina , Nanoestruturas/química , Poli A , Conformação Proteica em Folha beta , Água
12.
BMC Genomics ; 23(1): 530, 2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35869428

RESUMO

BACKGROUND: Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species. RESULTS: Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality. CONCLUSIONS: Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.


Assuntos
Poli A , RNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Poli A/genética , Poli A/metabolismo , Poliadenilação , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma
13.
Molecules ; 27(14)2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35889444

RESUMO

Cancer is a leading cause of mortality globally. Despite remarkable improvements in cancer-treatment approaches, disease recurrence and progression remain major obstacles to therapy. While chemotherapy is still a first-line treatment for a variety of cancers, the focus has shifted to the development and application of new approaches to therapy. Nevertheless, the relationship between immune response, neoplastic diseases and treatment efficiency is not fully understood. Therefore, the aim of the study was to investigate the immunopharmacological effects of methacrylic acid homopolymer in an in vivo tumor model. MATERIALS AND METHODS: Monomeric methacrylic acid was used to synthesize polymers. Methacrylic acid was polymerized in dioxane in the presence of 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid. To study the molecular weight characteristics of PMAA by GPC, carboxyl groups were preliminarily methylated with diazomethane. An experimental cancer model was obtained by grafting RMK1 breast cancer cells. The serum levels of IL-6, IL-10, IL-17, transforming growth factor ß1 (TGF-ß1), and tumor necrosis factor α (TNF-α) were measured by ELISA. RESULTS: The effect of PMAA on the serum concentrations of several cytokines was studied upon its single administration to laboratory animals in early neoplastic process. The IL-6, IL-17 and TGF-ß1 concentrations were found to change significantly and reach the level observed in intact rats. The IL-10 concentration tended to normalize. CONCLUSION: The positive results obtained are the basis for further studies on the effect of methacrylic-acid polymers with different molecular-weight characteristics on the neoplastic process.


Assuntos
Citocinas , Neoplasias Experimentais , Ácidos Polimetacrílicos , Animais , Interleucina-10 , Interleucina-17 , Interleucina-6 , Neoplasias Experimentais/tratamento farmacológico , Poli A , Polímeros , Ácidos Polimetacrílicos/farmacologia , Ratos , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa
14.
Biosens Bioelectron ; 214: 114497, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35797934

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The aberrant expression of miRNAs is related to many diseases. MiRNAs can serve as potential biomarkers for the prognosis and diagnosis of cancers and other human diseases. However, the short sequence and high sequence similarity of miRNAs impede detection. Herein, we propose a method to integrate polyA-tailing and CRISPR/Cas12a to amplify and detect all miRNAs with high specificity and sensitivity. PolyA-tailing enables efficient amplification of RNA and introduces a universal PAM sequence for Cas12a to unlock its PAM restriction. The CRISPR-Cas system guarantees the specific recognition of nucleic acid sequences with a single base mismatch. A limit of detection (LOD) as low as 50 fM was achieved. The practical application ability of polyA-CRISPR/Cas12a-based miRNA detection was validated by miRNA analyses in multiple cancer cell samples. With the increasing stability of RNA samples, low cost, excellent specificity, and sensitivity, this method demonstrates great potential to scale up to parallel diagnostic sets for miRNA-related disease.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Humanos , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Poli A/genética
15.
Nat Protoc ; 17(9): 1980-2007, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35831615

RESUMO

Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.


Assuntos
Poli A , Transcriptoma , Animais , DNA Complementar/genética , Mamíferos/genética , Camundongos , Poli A/genética , RNA , RNA Mensageiro/análise , Análise de Sequência de RNA/métodos
16.
Mikrochim Acta ; 189(7): 244, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35674802

RESUMO

A novel Apt-LFA has been established for kanamycin based on non-thiolated nucleic acid-modified colloidal gold nanoprobe (AuNPs@polyA-DNA). The improvement in nucleic acid hybridization speed and efficiency was verified by modifying AuNPs with polyA-DNA strands instead of thiolated oligonucleotides (SH-DNA) strands. Moreover, the AuNPs@polyA-DNA was explored to apply in an Apt-LFA. The experimental factors including the concentration of the aptamer, the concentration of SA-DNAT conjugate, the incubation time, and temperature were carefully investigated. In addition, the kanamycin aptamer was modified by extending several bases at its end to modulate the hybridization complementary strand, which was found to significantly improve the performance of Apt-LFA. Under optimal experimental conditions, the Apt-LFA can detect kanamycin in honey with a LOD of 250 ng mL-1 by the naked eyes. A linear range of 50-1250 ng mL-1 was obtained with a LOD of 15 ng mL-1 in honey by a portable reader. The Apt-LFA was successfully applied to the detection of kanamycin in honey with recoveries of 95.1-105.2%.


Assuntos
Aptâmeros de Nucleotídeos , Nanopartículas Metálicas , Ácidos Nucleicos , Aptâmeros de Nucleotídeos/genética , DNA , Ouro , Canamicina , Limite de Detecção , Poli A
17.
Talanta ; 247: 123600, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35659686

RESUMO

Monitoring the level of matrix metalloproteinase-9 (MMP-9) and inhibiting its expression is important for the diagnosis and treatment of various diseases. However, the analysis of MMP-9 is challenging owing to its very low content in the blood, especially at the early stages of diseases. Therefore, we developed an ultrasensitive and easy-to-use immunosensor based on a three-dimensional (3D) bioplatform for the determination of the total MMP-9 concentration in plasma. The used 3D bioplatform (G2 poly(amidoamine) dendrimer; PAMAM) improved the sensitivity of the determination by significantly expanding the surface area of the receptor layer. The antigen-antibody recognition process was controlled by quartz crystal microbalance with dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS). The effect of the orientation of antibody molecules in the sensing layer on the work parameters of the immunosensor was analyzed using unmodified PAMAM (PAMAM-NH2) and PAMAM functionalized with -COOH groups (PAMAM-COOH). The developed immunosensor based on PAMAM-NH2 was characterized by a lower detection limit (LOD = 2.0 pg⋅mL-1) and wider analytical range (1·10-4 - 5 µg⋅mL-1 for EIS and QCM-D) compared to PAMAM-COOH immunosensor (EIS: 1·10-4 - 0.5 µg⋅mL-1; QCM-D: 5·10-4 - 0.5 µg⋅mL-1). The functionality of the proposed device was verified in spiked plasma. The recoveries determined in commercial human and rat plasma and noncommercial rat plasma were very close to the value of 100% and in the range of 96-120% for Au/PAMAM-NH2/Ab and Au/PAMAM-COOH/Ab immunosensors, respectively. The designed analytical devices showed high selectivity and sensitivity without the use of any amplifiers such as metal nanoparticles or enzymes.


Assuntos
Técnicas Biossensoriais , Dendrímeros , Nanopartículas Metálicas , Animais , Técnicas Biossensoriais/métodos , Dendrímeros/química , Técnicas Eletroquímicas/métodos , Ouro/química , Imunoensaio/métodos , Limite de Detecção , Metaloproteinase 9 da Matriz , Nanopartículas Metálicas/química , Poli A , Poliaminas , Ratos
18.
Mol Cell ; 82(11): 1979-1980, 2022 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-35659324

RESUMO

Viegas et al. (2022) discover that in Trypanosoma brucei the poly(A) tails of the variant surface glycoprotein (VSG) transcripts are methylated, a mechanism that stabilizes these transcripts and ensures protection against the immune response in mammals.


Assuntos
Trypanosoma brucei brucei , Glicoproteínas Variantes de Superfície de Trypanosoma , Animais , Mamíferos , Glicoproteínas de Membrana , Poli A/genética , RNA Mensageiro/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
19.
Anal Chem ; 94(27): 9851-9855, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35758157

RESUMO

Ultrathin nanosheets of two-dimensional covalent organic frameworks covered a quartz nanopipette and then acted as a nanopore device for single-molecule DNA sensing. Our results showed that a single DNA homopolymer as short as 6 bases could be detected. The dwell times of 30-mer DNA homopolymers were obviously longer than the times of 10- or 6-mer ones. For different bases, poly(dA)6 showed the slowest transport speed (∼595 µs/base) compared with cytosine (∼355 µs/base) in poly(dC)6 and thymine (∼220 µs/base) in poly(dT)6. Such translocation speeds are the slowest ever reported in two-dimensional material-based nanopores. Poly(dA)6 also showed the biggest current blockade (94.74 pA) compared with poly(dC)6 (79.54 pA) and poly(dT)6 (71.41 pA). However, the present difference in blockade current was not big enough to distinguish the four DNA bases. Our study exhibits the shortest single DNA molecules that can be detected by COF nanopores at the present stage and lights the way for DNA sequencing based on solid-state nanopores.


Assuntos
Estruturas Metalorgânicas , Nanoporos , DNA , Nanotecnologia , Poli A , Análise de Sequência de DNA/métodos
20.
Sci Rep ; 12(1): 10599, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35732903

RESUMO

The full-length double-strand cDNA sequencing, one of the RNA-Seq methods, is a powerful method used to investigate the transcriptome status of a gene of interest, such as its transcription level and alternative splicing variants. Furthermore, full-length double-strand cDNA sequencing has the advantage that it can create a library from a small amount of sample and the library can be applied to long-read sequencers in addition to short-read sequencers. Nevertheless, one of our previous studies indicated that the full-length double-strand cDNA sequencing yields non-specific genomic DNA amplification, affecting transcriptome analysis, such as transcript quantification and alternative splicing analysis. In this study, it was confirmed that it is possible to produce the RNA-Seq library from only genomic DNA and that the full-length double-strand cDNA sequencing of genomic DNA yielded non-specific genomic DNA amplification. To avoid non-specific genomic DNA amplification, two methods were examined, which are the DNase I-treated full-length double-strand cDNA sequencing and poly(A) capture full-length double-strand cDNA sequencing. Contrary to expectations, the non-specific genomic DNA amplification was increased and the number of the detected expressing genes was reduced in DNase I-treated full-length double-strand cDNA sequencing. On the other hand, in the poly(A) capture full-length double-strand cDNA sequencing, the non-specific genomic DNA amplification was significantly reduced, accordingly the accuracy and the number of detected expressing genes and splicing events were increased. The expression pattern and percentage spliced in index of splicing events were highly correlated. Our results indicate that the poly(A) capture full-length double-strand cDNA sequencing improves transcript quantification accuracy and the detection ability of alternative splicing events. It is also expected to contribute to the determination of the significance of DNA variants to splicing events.


Assuntos
Processamento Alternativo , Poli A , DNA Complementar/genética , Desoxirribonuclease I/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Poli A/genética , Análise de Sequência de RNA/métodos , Transcriptoma
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