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1.
Nucleic Acids Res ; 44(3): e26, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26438536

RESUMO

Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3-5.2 % relative standard deviation. Excellent linearity was observed 0.99227-0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.


Assuntos
Perfilação da Expressão Gênica , Células-Tronco Neurais/metabolismo , RNA/genética , Transcriptoma , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Limite de Detecção , Processamento Pós-Transcricional do RNA , Espectrometria de Massas em Tandem/métodos
2.
Nucleic Acids Res ; 44(13): 6036-45, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27307604

RESUMO

Natural RNAs utilize extensive chemical modifications to diversify their structures and functions. 2-Thiouridine geranylation is a special hydrophobic tRNA modification that has been discovered very recently in several bacteria, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella Typhimurium The geranylated residues are located in the first anticodon position of tRNAs specific for lysine, glutamine and glutamic acid. This big hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. We aimed to systematically study the structure, function and biosynthesis mechanism of this geranylation pathway, as well as answer the question of why nature uses such a hydrophobic modification in hydrophilic RNA systems. Recently, we have synthesized the deoxy-analog of S-geranyluridine and showed the geranylated T-G pair is much stronger than the geranylated T-A pair and other mismatched pairs in the B-form DNA duplex context, which is consistent with the observation that the geranylated tRNA(Glu) UUC recognizes GAG more efficiently than GAA. In this manuscript we report the synthesis and base pairing specificity studies of geranylated RNA oligos. We also report extensive molecular simulation studies to explore the structural features of the geranyl group in the context of A-form RNA and its effect on codon-anticodon interaction during ribosome binding.


Assuntos
RNA de Transferência/genética , RNA/genética , Ribossomos/genética , Tiouridina/análogos & derivados , Anticódon/genética , Códon/genética , DNA de Forma B/genética , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Conformação de Ácido Nucleico , Biossíntese de Proteínas/genética , RNA/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Tiouridina/metabolismo
3.
Anal Chem ; 89(11): 5673-5677, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28474522

RESUMO

As DNA nanotechnology matures, there is increasing need for fast, reliable, and automated purification methods. Here, we develop UHPLC methods to purify self-assembled DNA nanoswitches, which are formed using DNA origami approaches and are designed to change conformations in response to a binding partner. We found that shear degradation hindered LC purification of the DNA nanoswitches, removing oligonucleotides from the scaffold strand and causing loss of function. However, proper choice of column, flow rate, and buffers enabled robust and automated purification of DNA nanoswitches without loss of function in under a half hour. Applying our approach to DNA origami structures, we found that ∼400 nm long nanotubes degraded under the gentlest flow conditions while ∼40 nm diameter nanospheres remained intact even under aggressive conditions. These examples show how fluid stresses can affect different DNA nanostructures during LC purification and suggest that shear forces may be relevant for some applications of DNA nanotechnology. Further development of this approach could lead to fast and automated purification of DNA nanostructures of various shapes and sizes, which would be an important advance for the field.


Assuntos
Cromatografia Líquida de Alta Pressão , DNA/química , Conformação de Ácido Nucleico , Resistência ao Cisalhamento , Cromatografia Líquida de Alta Pressão/métodos , DNA/isolamento & purificação , Nanoestruturas , Nanotecnologia , Nanotubos
4.
Chembiochem ; 18(1): 72-76, 2017 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-27805801

RESUMO

It was recently revealed that 5-methylcytosine (5mC) in mRNA, similar to its behavior in DNA, can be oxidized to produce 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), implying the potential regulatory roles of this post-transcriptional RNA modification. In this study, we demonstrate the in vitro oxidation of 5fC to 5-carboxycytidine (5caC) by the catalytic domain of mammalian ten-eleven translocation enzyme (TET1) in different RNA contexts. We observed that this oxidation process has very low sequence dependence and can take place in single-stranded, double-stranded, or hairpin forms of RNA sequences, although the overall conversion yields are low.


Assuntos
Citosina/análogos & derivados , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Sequência de Bases , Biocatálise , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Citosina/análise , Citosina/química , Citosina/metabolismo , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxirredução , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , RNA/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem
5.
J Neurosci ; 34(46): 15170-7, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25392485

RESUMO

Modified RNA molecules have recently been shown to regulate nervous system functions. This mini-review and associated mini-symposium provide an overview of the types and known functions of novel modified RNAs in the nervous system, including covalently modified RNAs, edited RNAs, and circular RNAs. We discuss basic molecular mechanisms involving RNA modifications as well as the impact of modified RNAs and their regulation on neuronal processes and disorders, including neural fate specification, intellectual disability, neurodegeneration, dopamine neuron function, and substance use disorders.


Assuntos
Encéfalo/metabolismo , RNA/química , RNA/metabolismo , Animais , Encéfalo/citologia , Encéfalo/patologia , Humanos , Metilação , Edição de RNA , RNA Circular , RNA não Traduzido/metabolismo , Receptores de Glutamato/metabolismo , Transtornos Relacionados ao Uso de Substâncias/genética , Transtornos Relacionados ao Uso de Substâncias/metabolismo
6.
Thorax ; 70(4): 320-5, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25661115

RESUMO

BACKGROUND: Healthcare associated infections, including ventilator associated pneumonia, are difficult to diagnose and treat, and are associated with significant morbidity, mortality and cost. We aimed to demonstrate proof of concept that breath volatile profiles were associated with the presence of clinically relevant pathogens in the lower respiratory tract. METHODS: Patients with sterile brain injury requiring intubation and ventilation on the intensive care unit were eligible for inclusion. Serial clinical and breath data were obtained three times a week, from admission up to a maximum of 10 days. Bronchial lavage for semiquantitative culture was collected immediately prior to breath sampling. Breath samples were collected in triplicate for off-line analysis by thermal-desorption/gas chromatography/time-of-flight mass spectrometry. Breath data were recorded as retention time/mass ion pairs, and analysed (pathogen present vs absent) by ANOVA-mean centre principal component analysis. RESULTS: Samples were collected from 46 patients (mean (SD) age 49 (19) years; 27 male). The dominant factors affecting breath sample analysis were the individual breath profile and duration of intubation. When these were taken into account, clear separation was seen between breath profiles at each time point by the presence/absence of pathogens. Loadings plots identified consistent metabolite peaks contributing to this separation at each time point. CONCLUSIONS: Breath volatile analysis is able to classify breath profiles of patients with and without significant pathogen load in the lower respiratory tract. If validated in independent cohorts, these findings could lead to development of rapid non-invasive point-of-care surveillance systems and diagnostics for lower respiratory tract infection in the intensive care unit.


Assuntos
Metabolômica/métodos , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Adulto , Idoso , Testes Respiratórios/métodos , Líquido da Lavagem Broncoalveolar/microbiologia , Estudos de Casos e Controles , Cuidados Críticos/métodos , Expiração , Feminino , Humanos , Unidades de Terapia Intensiva , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/microbiologia , Vigilância da População/métodos
7.
J Am Soc Mass Spectrom ; 33(3): 598-602, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35157447

RESUMO

Detection and characterization of cross-linked peptides of unknown chemical nature and location is challenging. An analytical workflow based on the use of 18O-labeling tryptic digestion ( Anal. Chem. 2013, 85, 5900-5908) was previously utilized to identify reduction-resistant scrambled disulfide dipeptides within an IgG that was exposed to light under forced degradation conditions ( Mol. Pharmaceutics 2018, 15, 1598-1606). The analytical workflow denoted as XChem-Finder, while effective, is cumbersome and requires extensive manual effort for detection of 18O-incorporated peptides and subsequent de novo sequencing of partial peptide sequences to aid in the identification of cross-linked peptides. Here, we provide an automatic workflow using Byos (Protein Metrics Inc.) to facilitate the detection of cross-linked peptides. The LC-MS/MS data files that were subjected to the XChem-Finder workflow that identified the scrambled disulfides were utilized as the test-case data set for the automated 18O-labeling workflow in Byos. The new workflow resulted in the detection of a photoinduced cross-linked dipeptide with unknown linker chemistry, which was subsequently identified as a cross-linked dipeptide with a novel cysteine-tryptophan (thioether) linkage. This work demonstrates that combining 18O-labeling tryptic digestion with the Byos workflow enables rapid detection of cross-linked dipeptides.


Assuntos
Dipeptídeos , Dissulfetos , Software , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Cisteína/química , Cisteína/metabolismo , Dipeptídeos/análise , Dipeptídeos/química , Dissulfetos/química , Dissulfetos/metabolismo , Triptofano/química , Triptofano/metabolismo , Fluxo de Trabalho
8.
J Am Soc Mass Spectrom ; 33(3): 510-520, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35157441

RESUMO

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.


Assuntos
Espectrometria de Massas/métodos , Oligorribonucleotídeos , Análise de Sequência/métodos , Elétrons , Oligorribonucleotídeos/análise , Oligorribonucleotídeos/química , Oligorribonucleotídeos/efeitos da radiação , Fotólise , Raios Ultravioleta
9.
ChemMedChem ; 14(7): 758-769, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30707489

RESUMO

The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 µg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4 µg mL-1 , the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd ≈24 µm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.


Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , RNA de Transferência/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Antibacterianos/química , Bactérias Gram-Positivas/genética , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , RNA Mensageiro/genética , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
10.
Chem Commun (Camb) ; 51(91): 16369-72, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26405057

RESUMO

The synthesis and base pairing of DNA duplexes containing the geranylated 2-thiothymidine have been investigated. This naturally existing hydrophobic modification could grant better base pairing stability to the T-G pair over normal T-A and other mismatched pairs in the duplex context. This study provides a potential explanation for the different codon recognition preferences of the geranylated tRNAs.


Assuntos
Pareamento de Bases , Oligodesoxirribonucleotídeos/química , Terpenos/síntese química , Timidina/análogos & derivados , Timidina/síntese química , Sequência de Bases , Simulação de Dinâmica Molecular , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química
11.
J Mass Spectrom ; 48(6): 703-12, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23722961

RESUMO

The challenges posed by the analysis of mono-nucleotide mixtures by direct infusion electrospray ionization were examined in the context of recent advances of mass spectrometry (MS) technologies. In particular, we evaluated the merits of high-resolution mass analysis, multistep gas-phase dissociation, and ion mobility determinations for the characterization of species with very similar or identical elemental composition. The high resolving power afforded by a linear trap quadrupole-orbitrap allowed the complete differentiation of overlapping isotopic distributions produced by nucleotides that differed by a single mass unit. Resolving (12)C signals from nearly overlapped (13)C contributions provided the exact masses necessary to calculate matching elemental compositions for unambiguous formulae assignment. However, it was the ability to perform sequential steps of gas-phase dissociation (i.e. MS(n)-type analysis) that proved more valuable for discriminating between truly isobaric nucleotides, such as the AMP/dGMP and UMP/ΨMP couples, which were differentiated in the mixture from their unique fragmentation patterns. The identification of diagnostic fragments enabled the deconvolution of dissociation spectra containing the products of coexisting isobars that could not be individually isolated in the mass-selection step. Approaches based on ion mobility spectrometry-MS provided another dimension upon which isobaric nucleotides could be differentiated according to their distinctive mobility behaviors. Subtle structural variations, such as the different positions of an oxygen atom in AMP/dGMP or the glycosidic bond in UMP/ΨMP, produced detectable differences in the respective ion mobility profiles, which enabled the differentiation of the isobaric couples in the mixture. Parallel activation of all ions emerging from the ion mobility element provided an additional dimension for differentiating these analytes on the basis of both mobility and fragmentation properties.


Assuntos
Nucleotídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Isótopos/química , Nucleotídeos/química , Nucleotídeos/isolamento & purificação , Espectrometria de Massas em Tandem
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