Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 87.109
Filtrar
1.
Anal Chim Acta ; 1182: 338946, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602192

RESUMO

This work reports the first electrochemical bioplatform developed for the multidetection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA, DNA N6-methyladenine (6mA) and RNA N6-methyladenosine (m6A) methylations at global level. Direct competitive immunoassays were implemented on the surface of magnetic beads (MBs) and optimized for the single amperometric determination of different targets varying in length, sequence and number of methylations on screen-printed carbon electrodes. After evaluating the sensitivity and selectivity of such determinations and the confirmation of no cross-reactivity, a multiplexed disposable platform allowing the simultaneous determination of the mentioned four methylation events in only 45 min has been prepared. The multiplexed bioplatform was successfully applied to the determination of m6A in cellular total RNA and of 5-mC, 5-hmC and 6mA in genomic DNA extracted from tissues. The developed bioplatform showed its usefulness to discriminate the aggressiveness of cancerous cells and between healthy and tumor tissues of colorectal cancer patients.


Assuntos
Ácidos Nucleicos , Adenosina , Humanos , Fenômenos Magnéticos , Metilação , RNA
2.
Life Sci Alliance ; 4(12)2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34593555

RESUMO

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.


Assuntos
COVID-19/virologia , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Técnicas Biossensoriais , COVID-19/diagnóstico , Humanos , Luminescência , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/genética , RNA Viral/genética , SARS-CoV-2/genética
3.
Curr Protoc ; 1(10): e270, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34619810

RESUMO

The ConSurf web server (https://consurf.tau.ac.il/) for using evolutionary data to detect functional regions is useful for analyzing proteins. The analysis is based on the premise that functional regions, which may for example facilitate ligand binding and catalysis, often evolve slowly. The analysis requires finding enough effective, i.e., non-redundant, sufficiently remote homologs. Indeed, the ConSurf pipeline, which is based on state-of-the-art protein sequence databases and analysis tools, is highly valuable for protein analysis. ConSurf also allows evolutionary analysis of RNA, but the analysis often fails due to insufficient data, particularly the inability of the current pipeline to detect enough effective RNA homologs. This is because the RNA search tools and databases offered are not as good as those used for protein analysis. Fortunately, ConSurf also allows importing external collections of homologs in the form of a multiple sequence alignment (MSA). Leveraging this, here we describe various protocols for constructing MSAs for successful ConSurf analysis of RNA queries. We report the level of success of these protocols on an exemplary set comprising a dozen RNA molecules of diverse structure and function. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Standard ConSurf evolutionary conservation analysis of an RNA query. Basic Protocol 2: ConSurf evolutionary conservation analysis of an RNA query with external MSA. Support Protocol 1: Construction of an MSA for an RNA query using other online servers. Support Protocol 2: Construction of an MSA for an RNA query using nHMMER locally.


Assuntos
Proteínas , RNA , Sequência Conservada , Bases de Dados de Proteínas , Proteínas/genética , Alinhamento de Sequência
4.
J Vis Exp ; (175)2021 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-34605806

RESUMO

RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in diagnostics, prognostics, and therapeutics of tumors. Differential analysis of RNA-seq data is crucial to identify aberrant transcriptions, and limma, EdgeR and DESeq2 are efficient tools for differential analysis. However, RNA-seq differential analysis requires certain skills with R language and the ability to choose an appropriate method, which is lacking in the curriculum of medical education. Herein, we provide the detailed protocol to identify differentially expressed genes (DEGs) between cholangiocarcinoma (CHOL) and normal tissues through limma, DESeq2 and EdgeR, respectively, and the results are shown in volcano plots and Venn diagrams. The three protocols of limma, DESeq2 and EdgeR are similar but have different steps among the processes of the analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Additionally, the normalized RNA-seq count data is necessary for EdgeR and limma but is not necessary for DESeq2. Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. The results of the three methods are partly overlapping. All three methods have their own advantages, and the choice of method only depends on the data.


Assuntos
Perfilação da Expressão Gênica , Software , RNA , Análise de Sequência de RNA , Transcriptoma
5.
J Vis Exp ; (175)2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34605813

RESUMO

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla. Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 °C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.


Assuntos
Núcleo Celular , Transcriptoma , Animais , Rim , Camundongos , RNA , Análise de Sequência de RNA
6.
Nat Commun ; 12(1): 5732, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593797

RESUMO

Although alterations in chromatin structure are known to exist in tumors, how these alterations relate to molecular phenotypes in cancer remains to be demonstrated. Multi-omics profiling of human tumors can provide insight into how alterations in chromatin structure are propagated through the pathway of gene expression to result in malignant protein expression. We applied multi-omics profiling of chromatin accessibility, RNA abundance, and protein abundance to 36 human thyroid cancer primary tumors, metastases, and patient-match normal tissue. Through quantification of chromatin accessibility associated with active transcription units and global protein expression, we identify a local chromatin structure that is highly correlated with coordinated RNA and protein expression. In particular, we identify enhancers located within gene-bodies as predictive of correlated RNA and protein expression, that is independent of overall transcriptional activity. To demonstrate the generalizability of these findings we also identify similar results in an independent cohort of human breast cancers. Taken together, these analyses suggest that local enhancers, rather than distal enhancers, are likely most predictive of cancer gene expression phenotypes. This allows for identification of potential targets for cancer therapeutic approaches and reinforces the utility of multi-omics profiling as a methodology to understand human disease.


Assuntos
Neoplasias da Mama/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Mama/patologia , Sequenciamento de Cromatina por Imunoprecipitação , Estudos de Coortes , Conjuntos de Dados como Assunto , Elementos Facilitadores Genéticos , Epigênese Genética , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Regiões Promotoras Genéticas , Proteômica , RNA/metabolismo , RNA-Seq , Câncer Papilífero da Tireoide/patologia , Câncer Papilífero da Tireoide/cirurgia , Glândula Tireoide/patologia , Glândula Tireoide/cirurgia , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , Tireoidectomia , Fatores de Transcrição/metabolismo
7.
J Vis Exp ; (175)2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34633367

RESUMO

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.


Assuntos
Fibrose Cística , Pseudomonas aeruginosa , Biofilmes , Expressão Gênica , Humanos , Pseudomonas aeruginosa/genética , RNA
8.
BMC Bioinformatics ; 22(1): 495, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645386

RESUMO

BACKGROUND: Circular RNA (circRNA) is an emerging class of RNA molecules attracting researchers due to its potential for serving as markers for diagnosis, prognosis, or therapeutic targets of cancer, cardiovascular, and autoimmune diseases. Current methods for detection of circRNA from RNA sequencing (RNA-seq) focus mostly on improving mapping quality of reads supporting the back-splicing junction (BSJ) of a circRNA to eliminate false positives (FPs). We show that mapping information alone often cannot predict if a BSJ-supporting read is derived from a true circRNA or not, thus increasing the rate of FP circRNAs. RESULTS: We have developed Circall, a novel circRNA detection method from RNA-seq. Circall controls the FPs using a robust multidimensional local false discovery rate method based on the length and expression of circRNAs. It is computationally highly efficient by using a quasi-mapping algorithm for fast and accurate RNA read alignments. We applied Circall on two simulated datasets and three experimental datasets of human cell-lines. The results show that Circall achieves high sensitivity and precision in the simulated data. In the experimental datasets it performs well against current leading methods. Circall is also substantially faster than the other methods, particularly for large datasets. CONCLUSIONS: With those better performances in the detection of circRNAs and in computational time, Circall facilitates the analyses of circRNAs in large numbers of samples. Circall is implemented in C++ and R, and available for use at https://www.meb.ki.se/sites/biostatwiki/circall and https://github.com/datngu/Circall.


Assuntos
RNA Circular , RNA , Humanos , RNA/genética , Splicing de RNA , RNA-Seq , Análise de Sequência de RNA
9.
BMC Bioinformatics ; 22(1): 504, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34656080

RESUMO

BACKGROUND: The functions of RNA molecules are mainly determined by their secondary structures. These functions can also be predicted using bioinformatic tools that enable the alignment of multiple RNAs to determine functional domains and/or classify RNA molecules into RNA families. However, the existing multiple RNA alignment tools, which use structural information, are slow in aligning long molecules and/or a large number of molecules. Therefore, a more rapid tool for multiple RNA alignment may improve the classification of known RNAs and help to reveal the functions of newly discovered RNAs. RESULTS: Here, we introduce an extremely fast Python-based tool called RNAlign2D. It converts RNA sequences to pseudo-amino acid sequences, which incorporate structural information, and uses a customizable scoring matrix to align these RNA molecules via the multiple protein sequence alignment tool MUSCLE. CONCLUSIONS: RNAlign2D produces accurate RNA alignments in a very short time. The pseudo-amino acid substitution matrix approach utilized in RNAlign2D is applicable for virtually all protein aligners.


Assuntos
RNA , Software , Algoritmos , Substituição de Aminoácidos , Humanos , Conformação de Ácido Nucleico , RNA/genética , Alinhamento de Sequência , Análise de Sequência de RNA
10.
J Phys Chem B ; 125(38): 10672-10681, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34524834

RESUMO

Understanding the dynamics of the SARS CoV-2 RNA genome and its dependence on temperature is necessary to fight the current COVID-19 crisis. Computationally, the handling of large data is a major challenge in the elucidation of the structures of RNA. This work presents network analysis as an important tool to see the conformational evolution and the most dominant structures of the RNA genome at six different temperatures. It effectively distinguished different communities of RNA having structural variation. It is found that at higher temperatures (348 K and above), 80% of the RNA structure is destroyed in both the SPC/E and mTIP3P water models. The thermal denaturation free energy change ΔΔG value calculated for the long-lived structure at higher temperatures of 348 and 363 K ranges from 2.58 to 2.78 kcal/mol for the SPC/E water model, which agrees well with the experimentally reported thermal denaturation free energy range of 2.874 kcal/mol of SARS CoV-NP at normal pH. At higher temperatures, the stability of RNA conformation is found to be due to the existence of non-native base pairs in the SPC/E water model.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Conformação de Ácido Nucleico , RNA , Temperatura
11.
Elife ; 102021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34581669

RESUMO

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.


Assuntos
Sequência de Bases , Coronavirus/genética , Genoma Viral , RNA , SARS-CoV-2/genética , COVID-19/virologia , DNA Complementar , Biblioteca Gênica , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nanoporos , Reação em Cadeia da Polimerase , RNA Mensageiro , RNA Viral/genética , Recombinação Genética , Sequenciamento Completo do Genoma
12.
Methods Enzymol ; 658: 137-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34517945

RESUMO

The past decade has seen an exponential increase in the identification of individual nucleobases that undergo base conversion and/or modification in transcriptomes. While the enzymes that catalyze these types of changes have been identified, the global interactome of these modifiers is still largely unknown. Furthermore, in some instances, redundancy among a family of enzymes leads to an inability to pinpoint the protein responsible for modifying a given transcript merely from high-throughput sequencing data. This chapter focuses on a method for global identification of transcripts recognized by an RNA modification/editing enzyme via capture of the RNAs that are bound in vivo, a method referred as RNA immunoprecipitation (RIP). We provide a guide of the major issues to consider when designing a RIP experiment, a detailed experimental protocol as well as troubleshooting advice. The RIP protocol presented here can be readily applied to any organism or cell line of interest as well as both RNA modification enzymes and RNA-binding proteins (RBPs) that regulate RNA modification levels. As mentioned at the end of the protocol, the RIP assay can be coupled to high-throughput sequencing to globally identify bound targets. For more quantitative investigations, such as how binding of an RNA modification enzyme/regulator to a given target changes during development/in specific tissues or assessing how the presence or absence of RNA modification affects transcript recognition by a particular RBP (irrespective of a role for the RBP in modulating modification levels); the RIP assay should be coupled to quantitative real-time PCR (qRT-PCR).


Assuntos
Edição de RNA , RNA , Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
13.
Comput Biol Med ; 137: 104792, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34478921

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in the global coronavirus disease 2019 (COVID-19) pandemic. Despite several single-cell RNA sequencing (RNA-seq) studies, conclusions cannot be reached owing to the small number of available samples and the differences in technology and tissue types used in the studies. To better understand the cellular landscape and disease severity in COVID-19, we performed a meta-analysis of publicly available single-cell RNA-seq data from peripheral blood and lung samples of COVID-19 patients with varying degrees of severity. Patients with severe disease showed increased numbers of M1 macrophages in lung tissue, while the number of M2 macrophages was depleted. Cellular profiling of the peripheral blood showed a marked increase of CD14+, CD16+ monocytes and a concomitant depletion of overall B cells and CD4+, CD8+ T cells in severe patients when compared with moderate patients. Our analysis indicates the presence of faulty innate-to-adaptive switching, marked by a prolonged innate immune response and a dysregulated adaptive immune response in severe COVID-19 patients. Furthermore, we identified cell types with a transcriptome signature that can be used as a prognostic biomarker for disease state prediction and the effective therapeutic management of COVID-19 patients.


Assuntos
COVID-19 , RNA , Linfócitos T CD8-Positivos , Humanos , SARS-CoV-2 , Análise de Sequência de RNA
14.
Anal Chem ; 93(39): 13196-13203, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34546711

RESUMO

Gene expression analysis (e.g., targeted gene panels and transcriptomics) from whole blood can elucidate mechanisms of the immune function and aid in the discovery of biomarkers. Conventional venipuncture offers only a small snapshot of our broad immune landscape as immune responses may occur outside of the time and location parameters available for conventional venipuncture. A self-operated method that enables flexible sampling of liquid whole blood coupled with immediate stabilization of cellular RNA is instrumental in facilitating capture and preservation of acute or transient immune fluxes. To this end, we developed homeRNA, a kit for self-collection of peripheral blood (∼0.5 mL) and immediate stabilization of cellular RNA, using the Tasso-SST blood collection device with a specially designed stabilizer tube containing RNAlater. To assess the feasibility of homeRNA for self-collection and stabilization of whole blood RNA, we conducted a pilot study (n = 47 participants) in which we sent homeRNA to participants aged 21-69, located across 10 US states (94% successful blood collections, n = 61/65). Among participants who successfully collected blood, 93% reported no or minimal pain/discomfort using the kit (n = 39/42), and 79% reported very easy/somewhat easy stabilization protocol (n = 33/42). Total RNA yield from the stabilized samples ranged between 0.20 and 5.99 µg (mean = 1.51 µg), and all but one RNA integrity number values were above 7.0 (mean = 8.1), indicating limited RNA degradation. The results from this study demonstrate the self-collection and RNA stabilization of whole blood with homeRNA by participants themselves in their own home.


Assuntos
RNA , Humanos , Projetos Piloto
15.
Top Curr Chem (Cham) ; 379(6): 39, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34590223

RESUMO

Bioorthogonal reactions are rapid, specific and high yield reactions that can be performed in in vivo microenvironments or simulated microenvironments. At present, the main biorthogonal reactions include Staudinger ligation, copper-catalyzed azide alkyne cycloaddition, strain-promoted [3 + 2] reaction, tetrazine ligation, metal-catalyzed coupling reaction and photo-induced biorthogonal reactions. To date, many reviews have reported that bioorthogonal reactions have been used widely as a powerful tool in the field of life sciences, such as in target recognition, drug discovery, drug activation, omics research, visualization of life processes or exogenous bacterial infection processes, signal transduction pathway research, chemical reaction dynamics analysis, disease diagnosis and treatment. In contrast, to date, few studies have investigated the application of bioorthogonal reactions in the analysis of biomacromolecules in vivo. Therefore, the application of bioorthogonal reactions in the analysis of proteins, nucleic acids, metabolites, enzyme activities and other endogenous molecules, and the determination of disease-related targets is reviewed. In addition, this review discusses the future development opportunities and challenges of biorthogonal reactions. This review presents an overview of recent advances for application in biomolecular analysis and disease diagnosis, with a focus on proteins, metabolites and RNA detection.


Assuntos
Neoplasias/diagnóstico , Proteínas/análise , RNA/análise , Biomarcadores/análise , Reação de Cicloadição , Fezes/química , Corantes Fluorescentes/química , Fumaratos/análise , Humanos , Proteínas/química , Proteínas/metabolismo , RNA/química
16.
Biochemistry (Mosc) ; 86(8): 913-925, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488569

RESUMO

Once it was believed that ribosomal RNA encodes proteins, and GTP hydrolysis supplies the energy for protein synthesis. Everything has changed, when Alexander Spirin joined the science. It turned out that proteins are encoded by a completely different RNA, and GTP hydrolysis only accelerates the process already provided with energy. It was Spirin who first put forward the idea of a Brownian ratchet and explained how and why molecular machines could arise in the RNA world.


Assuntos
Guanosina Trifosfato/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Bioquímica/história , Catálise , DNA Bacteriano/análise , RNA Polimerases Dirigidas por DNA/química , História do Século XX , Hidrólise , Modelos Moleculares , Dobramento de Proteína , RNA/biossíntese , Ribossomos/fisiologia , U.R.S.S.
17.
Biochemistry (Mosc) ; 86(8): 952-961, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488572

RESUMO

A-minor motifs are RNA tertiary structure motifs that generally involve a canonical base pair and an adenine base forming hydrogen bonds with the minor groove of the base pair. Such motifs are among the most common tertiary interactions in known RNA structures, comparable in number with the non-canonical base pairs. They are often found in functionally important regions of non-coding RNAs and, in particular, play a central role in protein synthesis. Here, we review local variations of the A-minor geometry and discuss difficulties associated with their annotation, as well as various structural contexts and common A-minor co-motifs, and diverse functions of A-minors in various processes in a living cell.


Assuntos
Conformação de Ácido Nucleico , RNA/química , Pareamento de Bases , Ligação de Hidrogênio , Modelos Moleculares , RNA Bacteriano/metabolismo , RNA Catalítico/química , Ribossomos , Software
18.
Biochemistry (Mosc) ; 86(8): 962-975, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488573

RESUMO

Discovered almost twenty years ago, riboswitches turned out to be one of the most common regulatory systems in bacteria, with representatives found in eukaryotes and archaea. Unlike many other regulatory elements, riboswitches are entirely composed of RNA and capable of modulating expression of genes by direct binding of small cellular molecules. While bacterial riboswitches had been initially thought to control production of enzymes and transporters associated with small organic molecules via feedback regulatory circuits, later findings identified riboswitches directing expression of a wide range of genes and responding to various classes of molecules, including ions, signaling molecules, and others. The 5'-untranslated mRNA regions host a vast majority of riboswitches, which modulate transcription or translation of downstream genes through conformational rearrangements in the ligand-sensing domains and adjacent expression-controlling platforms. Over years, the repertoire of regulatory mechanisms employed by riboswitches has greatly expanded; most recent studies have highlighted the importance of alternative mechanisms, such as RNA degradation, for the riboswitch-mediated genetic circuits. This review discusses the plethora of bacterial riboswitch mechanisms and illustrates how riboswitches utilize different features and approaches to elicit various regulatory responses.


Assuntos
Estabilidade de RNA , Riboswitch/fisiologia , Regiões 5' não Traduzidas , Bacillus subtilis , Bactérias/metabolismo , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Ligantes , Fases de Leitura Aberta , RNA/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais
19.
Biochemistry (Mosc) ; 86(8): 1025-1040, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488578

RESUMO

The review discusses differences between the specific protein interactions with single- and double-stranded RNA molecules using the data on the structure of RNA-protein complexes. Proteins interacting with the single-stranded RNAs form contacts with RNA bases, which ensures recognition of specific nucleotide sequences. Formation of such contacts with the double-stranded RNAs is hindered, so that the proteins recognize unique conformations of the RNA spatial structure and interact mainly with the RNA sugar-phosphate backbone.


Assuntos
RNA de Cadeia Dupla/química , RNA/química , Motivos de Aminoácidos , Sequência de Bases , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Dedos de Zinco
20.
Biochemistry (Mosc) ; 86(8): 1012-1024, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488577

RESUMO

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2'-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer - EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.


Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , RNA/química , Anticorpos Monoclonais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Citoplasma/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB , Glioblastoma/genética , Humanos , Concentração Inibidora 50 , Células MCF-7 , Microscopia de Fluorescência , Oligonucleotídeos/química , Medicina de Precisão , Transporte Proteico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...