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1.
Chemosphere ; 286(Pt 1): 131614, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34325257

RESUMO

Particulate matter (PM)-induced airway inflammation contributes to the development and exacerbation of chronic airway diseases. Circular RNA (circRNA) is a new class of non-coding RNA that participates in gene regulation in various respiratory diseases, but the regulatory role of circRNA in PM-induced airway inflammation has not been fully elucidated. In this study, we performed the human circRNA microarray to reveal differentially expressed circRNAs in PM-induced human bronchial epithelial cells (HBECs). A total of 176 upregulated and 15 downregulated circRNAs were identified. Of these, a new circRNA termed circTXNRD1 was upregulated by PM exposure in a dose- and time-dependent manner. Knockdown of circTXNRD1 significantly attenuated PM-induced expression of proinflammatory cytokine interleukin 6 (IL-6). CircRNA pull-down, dual-luciferase reporter assay and fluorescence in situ hybridization showed that circTXNRD1 acted as an endogenous sponge to decrease miR-892a levels in HBECs. Downregulation of miR-892a could increase cyclooxygenase-2 (COX-2) expression and eventually promote IL-6 secretion in PM-induced HBECs. Taken together, our findings reveal circTXNRD1 as a novel inflammatory mediator in PM-induced inflammation in HBECs via regulating miR-892a/COX-2 axis. These results provide new insight into the biological mechanism underlying PM-induced inflammation in chronic airway diseases.


Assuntos
MicroRNAs , RNA Circular , Ciclo-Oxigenase 2/genética , Células Epiteliais , Humanos , Hibridização in Situ Fluorescente , Inflamação/induzido quimicamente , Inflamação/genética , MicroRNAs/genética , Material Particulado/toxicidade , RNA/genética
2.
J Exp Med ; 219(1)2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34757384

RESUMO

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral infection in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I)-dependent manner. SLR14 demonstrated remarkable prophylactic protective capacity against lethal SARS-CoV-2 infection and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity in the absence of the adaptive immune system. In the context of infection with variants of concern (VOCs), SLR14 conferred broad protection against emerging VOCs. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and treatment of chronically infected immunosuppressed patients.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/metabolismo , Modelos Animais de Doenças , Imunidade Inata/efeitos dos fármacos , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
3.
Methods Mol Biol ; 2404: 43-52, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694602

RESUMO

The new flow of high-throughput RNA secondary structure data coming from different techniques allowed the further development of machine learning approaches. We developed CROSS and CROSSalive, two algorithms trained on experimental data able to predict the RNA secondary structure propensity both in vitro and in vivo. Since the in vivo folding of RNA molecules depends on multiple factors due to the cellular crowded environment, prediction is a complex problem that needs additional calculations for the interaction with proteins and other molecules. In the following chapter, we will describe the differences in predicting RNA secondary structure propensity using experimental data as input for an Artificial Neural Network (ANN) in vitro and in vivo.


Assuntos
Redes Neurais de Computação , Aprendizado de Máquina , Estrutura Secundária de Proteína , RNA/genética
4.
Methods Mol Biol ; 2404: 53-65, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694603

RESUMO

RNA-binding proteins (RBPs) play a key role in post-transcriptional regulation via binding to coding and non-coding RNAs. Recent development in experimental technologies, aimed to identify the targets of RBPs, has significantly broadened our knowledge on protein-RNA interactions. However, for many RBPs in many organisms and cell types, experimental RNA-binding data is not available. In this chapter we describe a computational approach, named RBPmap, available as a web service via http://rbpmap.technion.ac.il/ and as a stand-alone version for download. RBPmap was designed for mapping and predicting the binding sites of any RBP within a nucleic acid sequence, given the availability of an experimentally defined binding motif of the RBP. The algorithm searches for a sub-sequence that significantly matches the RBP motif, considering the clustering propensity of other weak matches within the motif environment. Here, we present different applications of RBPmap for discovering the involvement of RBPs and their targets in a variety of cellular processes, in health and disease states. Finally, we demonstrate the performance of RBPmap in predicting the binding targets of RBPs in large-scale RNA-binding data, reinforcing the strength of the tool in distinguishing cognate binding sites from weak motifs.


Assuntos
RNA/química , Algoritmos , Sítios de Ligação , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA
5.
Methods Mol Biol ; 2404: 111-133, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694606

RESUMO

CRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome. Here, we describe RIP-seq as a method to globally identify the direct RNA binding partners of CRISPR-Cas RNPs using the Cas9 nuclease as an example. RIP-seq combines co-immunoprecipitation (coIP) of an epitope-tagged Cas9 followed by isolation and deep sequencing analysis of its co-purified bound RNAs. This method can not only be used to study interactions of Cas9 with its known interaction partners, crRNAs and tracrRNA in native systems, but also to reveal potential additional RNA substrates of Cas9. For example, in RIP-seq analysis of Cas9 from the foodborne pathogen Campylobacter jejuni (CjeCas9), we recently identified several endogenous RNAs bound to CjeCas9 RNP in a crRNA-dependent manner, leading to the discovery of PAM-independent RNA cleavage activity of CjeCas9 as well as non-canonical crRNAs. RIP-seq can be easily adapted to any other effector RNP of choice from other CRISPR-Cas systems, allowing for the identification of target RNAs. Deciphering novel RNA-protein interactions for CRISPR-Cas proteins within host bacterial genomes will lead to a better understanding of the molecular mechanisms and functions of these systems and enable us to use the in vivo identified interaction rules as design principles for nucleic acid-targeting applications, fitted to each nuclease of interest.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas/genética , DNA , Endonucleases , RNA/genética , RNA Guia/genética
6.
Methods Mol Biol ; 2404: 135-153, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694607

RESUMO

Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.


Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Sistema Livre de Células/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , RNA
7.
Methods Mol Biol ; 2404: 157-165, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694608

RESUMO

RNA is never left alone throughout its life cycle. Together with proteins, RNAs form membraneless organelles, called ribonucleoprotein particles (RNPs) where these two types of macromolecules strongly influence each other's functions and destinies. RNA immunoprecipitation is still one of the favorite techniques which allows to simultaneously study both the RNA and protein composition of the RNP complex.


Assuntos
RNA/genética , Imunoprecipitação
8.
Methods Mol Biol ; 2404: 167-188, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694609

RESUMO

During post-transcriptional gene regulation (PTGR), RNA binding proteins (RBPs) interact with all classes of RNA to control RNA maturation, stability, transport, and translation. Here, we describe Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a transcriptome-scale method for identifying RBP binding sites on target RNAs with nucleotide-level resolution. This method is readily applicable to any protein directly contacting RNA, including RBPs that are predicted to bind in a sequence- or structure-dependent manner at discrete RNA recognition elements (RREs), and those that are thought to bind transiently, such as RNA polymerases or helicases.


Assuntos
Transcriptoma , Sítios de Ligação , Imunoprecipitação , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleosídeos
9.
Methods Mol Biol ; 2404: 189-205, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34694610

RESUMO

Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) sequencing and its derivative enhanced CLIP (eCLIP) sequencing are methods for the transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). This chapter provides a stepwise tutorial for analyzing iCLIP and eCLIP data with replicates and size-matched input (SMI) controls after read alignment using our open-source tools htseq-clip and DEWSeq. This includes the preparation of gene annotation, extraction, and preprocessing of truncation sites and the detection of significantly enriched binding sites using a sliding window based approach suitable for different binding modes of RBPs.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Sítios de Ligação , Imunoprecipitação , RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma
10.
Environ Pollut ; 292(Pt B): 118446, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34737027

RESUMO

DNA metabarcoding can provide a high-throughput and rapid method for characterizing responses of communities to environmental stressors. However, within bulk samples, DNA metabarcoding hardly distinguishes live from the dead organisms. Here, both DNA and RNA metabarcoding were applied and compared in experimental freshwater mesocosms conducted for assessment of ecotoxicological responses of zooplankton communities to remediation treatment until 38 days post oil-spill. Furthermore, a novel indicator of normalized vitality (NV), sequence counts of RNA metabarcoding normalized by that of DNA metabarcoding, was developed for assessment of ecological responses. DNA and RNA metabarcoding detected similar taxa richness and rank of relative abundances. Both DNA and RNA metabarcoding demonstrated slight shifts in measured α-diversities in response to treatments. NV presented relatively greater magnitudes of differential responses of community compositions to treatments compared to DNA or RNA metabarcoding. NV declined from the start of the experiment (3 days pre-spill) to the end (38 days post-spill). NV also differed between Rotifer and Arthropoda, possibly due to differential life histories and sizes of organisms. NV could be a useful indicator for characterizing ecological responses to anthropogenic influence; however, the biology of target organisms and subsequent RNA production need to be considered.


Assuntos
Poluição por Petróleo , Zooplâncton , Animais , Biodiversidade , Código de Barras de DNA Taxonômico , Ecossistema , Água Doce , RNA
11.
BMC Genomics ; 22(Suppl 3): 787, 2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34727867

RESUMO

BACKGROUND: A new class of regulatory elements called super-enhancers, comprised of multiple neighboring enhancers, have recently been reported to be the key transcriptional drivers of cellular, developmental, and disease states. RESULTS: Here, we defined super-enhancer RNAs as highly expressed enhancer RNAs that are transcribed from a cluster of localized genomic regions. Using the cap analysis of gene expression sequencing data from FANTOM5, we systematically explored the enhancer and messenger RNA landscapes in hundreds of different cell types in response to various environments. Applying non-negative matrix factorization (NMF) to super-enhancer RNA profiles, we found that different cell types were well classified. In addition, through the NMF of individual time-course profiles from a single cell-type, super-enhancer RNAs were clustered into several states with progressive patterns. We further investigated the enriched biological functions of the proximal genes involved in each pattern, and found that they were associated with the corresponding developmental process. CONCLUSIONS: The proposed super-enhancer RNAs can act as a good alternative, without the complicated measurement of histone modifications, for identifying important regulatory elements of cell type specification and identifying dynamic cell states.


Assuntos
Elementos Facilitadores Genéticos , RNA , Diferenciação Celular , Elementos Facilitadores Genéticos/genética , RNA/genética , RNA Mensageiro/genética
13.
Molecules ; 26(21)2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34771026

RESUMO

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.


Assuntos
COVID-19/diagnóstico , COVID-19/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Teste para COVID-19/métodos , Humanos , Programas de Rastreamento/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/isolamento & purificação , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Saliva/química , Sensibilidade e Especificidade , Manejo de Espécimes/métodos
14.
BMC Bioinformatics ; 22(1): 554, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34781902

RESUMO

BACKGROUND: Protein-RNA interactions play key roles in many processes regulating gene expression. To understand the underlying binding preference, ultraviolet cross-linking and immunoprecipitation (CLIP)-based methods have been used to identify the binding sites for hundreds of RNA-binding proteins (RBPs) in vivo. Using these large-scale experimental data to infer RNA binding preference and predict missing binding sites has become a great challenge. Some existing deep-learning models have demonstrated high prediction accuracy for individual RBPs. However, it remains difficult to avoid significant bias due to the experimental protocol. The DeepRiPe method was recently developed to solve this problem via introducing multi-task or multi-label learning into this field. However, this method has not reached an ideal level of prediction power due to the weak neural network architecture. RESULTS: Compared to the DeepRiPe approach, our Multi-resBind method demonstrated substantial improvements using the same large-scale PAR-CLIP dataset with respect to an increase in the area under the receiver operating characteristic curve and average precision. We conducted extensive experiments to evaluate the impact of various types of input data on the final prediction accuracy. The same approach was used to evaluate the effect of loss functions. Finally, a modified integrated gradient was employed to generate attribution maps. The patterns disentangled from relative contributions according to context offer biological insights into the underlying mechanism of protein-RNA interactions. CONCLUSIONS: Here, we propose Multi-resBind as a new multi-label deep-learning approach to infer protein-RNA binding preferences and predict novel interactions. The results clearly demonstrate that Multi-resBind is a promising tool to predict unknown binding sites in vivo and gain biology insights into why the neural network makes a given prediction.


Assuntos
Proteínas de Ligação a RNA , RNA , Sítios de Ligação , Redes Neurais de Computação , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
15.
Anal Chem ; 93(45): 14907-14911, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34735132

RESUMO

Exosomes are nanosized extracellular vesicles that have a critical role in intercellular communication and tumor microenvironment regulation. Extensive research has shown that exosomal small RNAs contribute to metastasis in multiple tumor types and that abnormal epigenetic modifications in nucleic acids also have an association with diverse diseases. However, the content of modified nucleosides on exosomal small RNAs has not been quantitatively reported. Because of the trace amounts of exosomes and matrix complexity, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a powerful tool for label-free sensitive and simultaneous determinations of six important modified nucleosides on small RNAs inside exosomes. This system performed well using only approximately 107-108 particles of exosomes to obtain modified nucleoside levels between 0.001 and 0.03, and the most striking result was that the content of m6A in exosomal small RNAs was continuously higher than that in the cells being analyzed. We hope that this conclusion helps establish a greater degree of deciphering accuracy on exosomes, which has considerable application potential in the diagnosis and prognosis of diseases.


Assuntos
Exossomos , RNA , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Epigênese Genética , Exossomos/genética , Espectrometria de Massas em Tandem
16.
Curr Protoc ; 1(11): e307, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34792865

RESUMO

This protocol describes a step-by-step chemical synthesis approach to prepare N3 -methylcytidine (m3 C) and its phosphoramidite. The method for synthesizing m3 C starts from commercially available cytidine, and proceeds via N3 -methylation in the presence of MeI, which generates the N3 -methylcytidine (m3 C) nucleoside, followed by the installation of several protecting groups at sites that include the 5'-hydroxyl group (4,4'-dimethoxytrityl protection), the 4-amino group (benzoyl protection), and the 2'-hydroxyl group (tert-butyldimethylsilyl, TBDMS, protection). Standard phosphoramidite chemistry is applied to prepare the final m3 C phosphoramidite for solid-phase synthesis of a series of RNA oligonucleotides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of N3 -methylcytidine (m3 C) and its phosphoramidite Basic Protocol 2: Automated synthesis of m3 C modified RNA oligonucleotides.


Assuntos
Oligonucleotídeos , RNA , Citidina , Nucleosídeos , Técnicas de Síntese em Fase Sólida
17.
BMC Res Notes ; 14(1): 418, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34794498

RESUMO

OBJECTIVE: Transcriptional profiling of immune cells is an indispensable tool in biomedical research; however, heterogenous sample types routinely used in transcriptomic studies may mask important cell type-specific transcriptional differences. Techniques to isolate desired cell types are used to overcome this limitation. We sought to evaluate the use of immunomagnetic B cell isolation on RNA quality and transcriptional output. Additionally, we aimed to develop a B cell gene signature representative of a freshly isolated B cell population to be used as a tool to verify isolation efficacy and to provide a transcriptional standard for evaluating maintenance or deviation from traditional B cell identity. RESULTS: We found RNA quality and RNA-sequencing output to be comparable between donor-matched PBMC, whole blood, and B cells following negative selection by immunomagnetic B cell isolation. Transcriptional analysis enabled the development of an 85 gene B cell signature. This signature effectively clustered isolated B cells from heterogeneous sample types in our study and naïve and memory B cells when applied to transcriptional data from a published source. Additionally, by identifying B cell signature genes whose functional role in B cells is currently unknown, our gene signature has uncovered areas for future investigation.


Assuntos
Linfócitos B , Leucócitos Mononucleares , Separação Celular , Perfilação da Expressão Gênica , RNA , Transcriptoma
18.
Curr Protoc ; 1(11): e299, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34826343

RESUMO

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule fluorescence in situ hybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in-tube approach for antibody staining that negates freeze-cracking, a validated method to perform the cost-reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sequential immunofluorescence and single-molecule fluorescence in situ hybridization Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single-molecule fluorescence in situ hybridization Basic Protocol 2: Simplified immunofluorescence in C. elegans embryos Basic Protocol 3: Single-molecule fluorescence in situ hybridization or single-molecule inexpensive fluorescence in situ hybridization.


Assuntos
Caenorhabditis elegans , RNA , Animais , Caenorhabditis elegans/genética , Imunofluorescência , Hibridização in Situ Fluorescente , RNA Mensageiro/genética
19.
Mol Biol (Mosk) ; 55(6): 1030-1044, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34837707

RESUMO

The development of approaches for predictive calculation of hybridization properties of various nucleic acid (NA) derivatives is the basis for the rational design of the NA-based constructs. Modern advances in computer modeling methods provide the feasibility of these calculations. We have analyzed the possibility of calculating the energy of DNA/RNA and RNA/RNA duplex formation using representative sets of complexes (65 and 75 complexes, respectively). We used the classical molecular dynamics (MD) method, the MMPBSA or MMGBSA approaches to calculate the enthalpy (ΔH°) component, and the quasi-harmonic approximation (Q-Harm) or the normal mode analysis (NMA) methods to calculate the entropy (ΔS°) contribution to the Gibbs energy (ΔG°37) of the NA complex formation. We have found that the MMGBSA method in the analysis of the MD trajectory of only the NA duplex and the empirical linear approximation allow calculation of the enthalpy of formation of the DNA, RNA, and hybrid duplexes of various lengths and GC content with an accuracy of 8.6%. Within each type of complex, the combination of rather efficient MMGBSA and Q-Harm approaches being applied to the trajectory of only the bimolecular complex makes it possible to calculate the ΔG°37 of the duplex formation with an error value of 10%. The high accuracy of predictive calculation for different types of natural complexes (DNA/RNA, DNA/RNA, and RNA/RNA) indicates the possibility of extending the considered approach to analogs and derivatives of nucleic acids, which gives a fundamental opportunity in the future to perform rational design of new types of NA-targeted sequence-specific compounds.


Assuntos
Simulação de Dinâmica Molecular , RNA , DNA , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA/genética , Termodinâmica
20.
BMC Bioinformatics ; 22(1): 557, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34798805

RESUMO

BACKGROUND: The research landscape of single-cell and single-nuclei RNA-sequencing is evolving rapidly. In particular, the area for the detection of rare cells was highly facilitated by this technology. However, an automated, unbiased, and accurate annotation of rare subpopulations is challenging. Once rare cells are identified in one dataset, it is usually necessary to generate further specific datasets to enrich the analysis (e.g., with samples from other tissues). From a machine learning perspective, the challenge arises from the fact that rare-cell subpopulations constitute an imbalanced classification problem. We here introduce a Machine Learning (ML)-based oversampling method that uses gene expression counts of already identified rare cells as an input to generate synthetic cells to then identify similar (rare) cells in other publicly available experiments. We utilize single-cell synthetic oversampling (sc-SynO), which is based on the Localized Random Affine Shadowsampling (LoRAS) algorithm. The algorithm corrects for the overall imbalance ratio of the minority and majority class. RESULTS: We demonstrate the effectiveness of our method for three independent use cases, each consisting of already published datasets. The first use case identifies cardiac glial cells in snRNA-Seq data (17 nuclei out of 8635). This use case was designed to take a larger imbalance ratio (~1 to 500) into account and only uses single-nuclei data. The second use case was designed to jointly use snRNA-Seq data and scRNA-Seq on a lower imbalance ratio (~1 to 26) for the training step to likewise investigate the potential of the algorithm to consider both single-cell capture procedures and the impact of "less" rare-cell types. The third dataset refers to the murine data of the Allen Brain Atlas, including more than 1 million cells. For validation purposes only, all datasets have also been analyzed traditionally using common data analysis approaches, such as the Seurat workflow. CONCLUSIONS: In comparison to baseline testing without oversampling, our approach identifies rare-cells with a robust precision-recall balance, including a high accuracy and low false positive detection rate. A practical benefit of our algorithm is that it can be readily implemented in other and existing workflows. The code basis in R and Python is publicly available at FairdomHub, as well as GitHub, and can easily be transferred to identify other rare-cell types.


Assuntos
RNA , Análise de Célula Única , Animais , Análise por Conglomerados , Aprendizado de Máquina , Camundongos , RNA/genética , Análise de Sequência de RNA
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