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1.
Mol Cell Neurosci ; 100: 103401, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31491533

RESUMO

Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.

2.
Methods Mol Biol ; 2035: 407-433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31444766

RESUMO

G-quadruplex structures have been suggested to be biologically important in processes such as transcription and translation, gene expression and regulation in human cancer cells, and regulation of telomere length. Investigation of G-quadruplex structures associated with biological events is therefore essential to understanding the functions of these molecules. We developed the 19F-labeled nucleobases and introduced them into DNA sequences for the 19F NMR spectroscopy analysis. We present the 19F NMR methodology used in our research group for the study of G-quadruplex structures in vitro and in living cells.

3.
Bioorg Med Chem ; 27(2): 364-369, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30545733

RESUMO

We synthesized several DNA oligonucleotides containing one or several 2'-O-methyl-8-methyl guanosine (m8Gm) and demonstrated that these oligonucleotides not only stabilize the Z-DNA with a wide range of sequences under low salt conditions but also possess high thermal stability. Using artificial nucleobase-containing oligonucleotides, we studied the interaction of the Zα domain with Z-DNA. Furthermore, we showed that the m8Gm-contained oligonucleotides allow to study the photochemical reaction of Z-DNA.


Assuntos
DNA Forma Z/química , Guanosina/análogos & derivados , Guanosina/química , Oligodesoxirribonucleotídeos/química , DNA Forma Z/síntese química , DNA Forma Z/metabolismo , DNA Forma Z/efeitos da radiação , Proteínas de Fluorescência Verde/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/efeitos da radiação , Oxirredução , Ligação Proteica , Temperatura de Transição , Raios Ultravioleta
4.
Molecules ; 23(10)2018 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-30304782

RESUMO

In contrast to Z-DNA that was stabilized and well-studied for its structure by chemical approaches, the stabilization and structural study of Z-RNA remains a challenge. In this study, we developed a Z-form RNA stabilizer m8Gm, and demonstrated that incorporation of m8Gm into RNA can markedly stabilize the Z-RNA at low salt conditions. Using the m8Gm-contained Z-RNA, we determined the structure of Z-RNA and investigated the interaction of protein and Z-RNA.

5.
Analyst ; 142(21): 4083-4088, 2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-28932835

RESUMO

In the present study, we developed a multi-functional guanine derivative, 8FG, as a G-quadruplex stabilizer, a fluorescent probe for the detection of G-quadruplex formation, and a 19F sensor for the observation of the G-quadruplex. We demonstrate that the functional nucleoside bearing a 3,5-bis(trifluoromethyl)benzene group at the 8-position of guanine stabilizes the DNA G-quadruplex structure and fluoresces following the G-quadruplex formation. Furthermore, we show that the functional sensor can be used to directly observe DNA G-quadruplexes by 19F-NMR in living cells. To our knowledge, this is the first study showing that the nucleoside derivative simultaneously allows for three kinds of functions at a single G-quadruplex DNA. Our results suggest that the multi-functional nucleoside derivative can be broadly used for studying the G-quadruplex structure and serves as a powerful tool for examining the molecular basis of G-quadruplex formation in vitro and in living cells.

6.
Sci Rep ; 7(1): 6695, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28751647

RESUMO

In this study, by combining nuclear magnetic resonance (NMR), circular dichroism (CD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and gel electrophoresis, we report an unusual topological structure of the RNA G-quadruplex motif formed by human telomere RNA r(UAGGGU) containing 8-bromoguanosine. Results showed that the RNA sequence formed an antiparallel tetramolecular G-quadruplex, in which each pair of diagonal strands run in opposite directions. Furthermore, guanosines were observed both in syn- and anti-conformations. In addition, two of these G-quadruplex subunits were found to be stacking on top of each other, forming a dimeric RNA G-quadruplex. Our findings provide a new insight into the behavior of RNA G-quadruplex structures.

7.
J Am Chem Soc ; 139(22): 7533-7539, 2017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28510424

RESUMO

Telomeric repeat-containing RNA is a new noncoding RNA molecule that performs various biofunctions. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an RNA-binding protein involved in the telomere maintenance machinery. To date, little is known about how hnRNPA1 binds to telomeric RNA. In this study, we investigated the binding affinity and recognition mechanism of telomere RNA with the RNA recognition motif of hnRNPA1. Using the photochemical cross-linking method, we showed that the telomere RNA G-quadruplex with loops is important in the interaction of telomere RNA with hnRNPA1. Using small-molecule probes, we directly visualized the complex formed by the telomere RNA G-quadruplex and hnRNPA1 in vitro and in live cells. The results suggested that the structure-dependent binding of hnRNPA1 to telomere RNA regulates the telomere function. Therefore, our study provides new insights into the interactions between the RNA G-quadruplex and proteins at the telomere.

8.
Nucleic Acids Res ; 45(9): 5501-5511, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28180296

RESUMO

Human telomeric RNA has been identified as a key component of the telomere machinery. Recently, the growing evidence suggests that the telomeric RNA forms G-quadruplex structures to play an important role in telomere protection and regulation. In the present studies, we developed a 19F NMR spectroscopy method to investigate the telomeric RNA G-quadruplex structures in vitro and in living cells. We demonstrated that the simplicity and sensitivity of 19F NMR approach can be used to directly observe the dimeric and two-subunits stacked G-quadruplexes in vitro and in living cells and quantitatively characterize the thermodynamic properties of the G-quadruplexes. By employing the 19F NMR in living cell experiment, we confirmed for the first time that the higher-order G-quadruplex exists in cells. We further demonstrated that telomere RNA G-quadruplexes are converted to the higher-order G-quadruplex under molecular crowding condition, a cell-like environment. We also show that the higher-order G-quadruplex has high thermal stability in crowded solutions. The finding provides new insight into the structural behavior of telomere RNA G-quadruplex in living cells. These results open new avenues for the investigation of G-quadruplex structures in vitro and in living cells.


Assuntos
Quadruplex G , Espectroscopia de Ressonância Magnética , RNA/química , Telômero/química , Animais , Sequência de Bases , Sobrevivência Celular , Flúor , Humanos , Substâncias Macromoleculares/metabolismo , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Temperatura Ambiente , Termodinâmica , Xenopus laevis
9.
J Am Chem Soc ; 139(7): 2565-2568, 2017 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-28177615

RESUMO

Human telomeric RNA performs various cellular functions such as telomere length regulation, heterochromatin formation, and chromosome end protection. Using a combination of nuclear magnetic resonance, circular dichroism, and gel electrophoresis, we observed an unusual topological structure formed by human telomere RNA r(GUUAGGGU). Our results showed that every set of four strands formed a parallel G-quadruplex as symmetry-related units containing four G-tetrads, two U-tetrads, and one A-tetrad. An eight-stranded helical fragment containing A-, G-, and U-tetrads provided a central intercalated scaffold that connected two G-quadruplex units in an alternating antiparallel arrangement, giving rise to a novel RNA architecture. This higher order RNA structure is so stable that it would be surprising if similar structures do not occur in nature. Our findings provide a new insight into the behavior of human telomeric RNA molecules.


Assuntos
Adenina/química , Quadruplex G , RNA/química , Telômero/química , Uridina/química , Humanos , Modelos Moleculares , Estabilidade de RNA
10.
ACS Omega ; 2(12): 8843-8848, 2017 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-30023592

RESUMO

In this study, we demonstrated that 19F NMR can be used to study the thrombin-binding aptamer (TBA) DNA G-quadruplex, widely used as a model structure for studying G-quadruplex aptamers. We systematically examined the structural feature of the TBA G-quadruplex aptamer with fluorine-19 (19F) labels at all of the thymidine positions. We successfully observed the structural change between the G-quadruplex and the unstructured single strand by 19F NMR spectroscopy. The thermodynamic parameters of these DNA G-quadruplex aptamers were also determined from the 19F NMR signals. We further showed that the 19F NMR method can be used to observe the complex formed by TBA G-quadruplex and thrombin. Our results suggest that 19F NMR spectroscopy is a useful approach to study the aptamer G-quadruplex structure.

11.
Sci Rep ; 6: 33217, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27620982

RESUMO

Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics.


Assuntos
Química Click/métodos , DNA/genética , Hibridização in Situ Fluorescente/métodos , Telômero/genética , Divisão Celular/genética , DNA/química , Diagnóstico por Imagem/métodos , Fluorescência , Corantes Fluorescentes/química , Células HeLa , Humanos , Reprodutibilidade dos Testes
12.
Curr Protoc Nucleic Acid Chem ; 61: 6.13.1-13, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-26344230

RESUMO

The detection of individual telomere lengths of human chromosomes can provide crucial information on genome stability, cancer, and telomere-related diseases. However, current methods to measure telomere length entail shortcomings that have limited their use. Recently, we have developed a method for detection of individual telomere lengths (DITL) that uses a chemistry-based DNA-cutting approach. The most beneficial feature of the DITL approach is to cleave the sequence adjacent to the telomere followed by resolution of the telomere length at the nucleotide level of a single chromosome. In this unit, a protocol for successful detection of individual telomere lengths from individual chromosomes is described in detail.


Assuntos
Ácidos Nucleicos Peptídicos/química , Biotecnologia/métodos , Cromossomos Humanos/genética , Humanos , Telômero/genética
13.
Bioorg Med Chem ; 22(16): 4419-21, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24947481

RESUMO

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA-RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA-RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA-RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA-RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA-RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.


Assuntos
Química Click , DNA/química , Quadruplex G , RNA/química , Telômero/química , Humanos , Conformação de Ácido Nucleico
14.
Angew Chem Int Ed Engl ; 52(51): 13681-4, 2013 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-24155125

RESUMO

Cut loose: A pseudocomplementary peptide nucleic acid was tethered to a pyrrole/imidazole hairpin polyamide, and was used to selectively target a specific DNA sequence. Binding even occurs under high salt conditions. Furthermore, the conjugate facilitated sequence-specific scission of long dsDNA. This simple approach promises to resolve the technical difficulties in targeting DNA sequences with PNA.


Assuntos
Amidas/química , DNA/química , Imidazóis/química , Ácidos Nucleicos Peptídicos/química , Pirróis/química , DNA/metabolismo , Clivagem do DNA
15.
J Am Chem Soc ; 135(1): 14-7, 2013 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-23252341

RESUMO

The understanding of telomeres is expected to provide major insights into genome stability, cancer, and telomere-related diseases. In recent years, there have been considerable improvements in the technologies available to determine the length of telomeres of human chromosomes; however, the present methods for measuring telomere length are fraught with shortcomings that have limited their use. Here we describe a method for detection of individual telomere lengths (DITL) that uses a chemistry-based approach that accurately measures the telomere lengths from individual chromosomes. The method was successfully used to determine telomere DNA by breaking in the target sequence and producing a "real telomere fragment." The DITL approach involves cleavage of the sequence adjacent to the telomere followed by resolution of the telomere length at the nucleotide level of a single chromosome. Comparison of the DITL method and the traditional terminal restriction fragment (TRF) analysis indicates that the DITL approach appears to be promising for the quantification of telomere repeats in each chromosome and the detection of accurate telomere lengths that can be missed using TRF analysis.


Assuntos
Cromossomos/química , DNA/química , Telômero/química , Células Cultivadas , Cromossomos/genética , DNA/genética , Células HEK293 , Células HeLa , Humanos , Telômero/genética
16.
Chem Commun (Camb) ; 48(88): 10835-7, 2012 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-23032097

RESUMO

Toward new biotechnology by genetic alphabet expansion, we developed an efficient site-specific labeling method for large RNA molecules. The combination of unnatural base pair transcription and post-transcriptional modification by click chemistry enables simple RNA labeling with a wide variety of functional groups at desired positions, in a one-pot reaction.


Assuntos
RNA/química , Pareamento de Bases , Química Click , Imidazóis/química , Piridinas/química , Pirróis/química , Processamento Pós-Transcricional do RNA , Transcrição Genética
17.
J Biol Chem ; 287(50): 41787-96, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23012368

RESUMO

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.


Assuntos
DNA/química , Oligonucleotídeos/química , RNA/química , Telômero/química , DNA/metabolismo , Humanos , Oligonucleotídeos/metabolismo , RNA/metabolismo , Telômero/metabolismo
18.
Angew Chem Int Ed Engl ; 51(29): 7198-202, 2012 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-22700182

RESUMO

Hole in one: A single peptide nucleic acid (PNA) effectively targets the G-rich region in double-stranded DNA through formation of a PNA/DNA hybrid G-quadruplex. Only one target site in the whole human genome was selectively cleaved by the hybrid G-quadruplex. Such site-selective scission of DNA is central to gene manipulation for molecular biology, biotechnology, and therapy.


Assuntos
DNA/química , Quadruplex G , Ácidos Nucleicos Peptídicos/química , Sequência de Bases , DNA/genética , DNA/metabolismo , Quebras de DNA , Genoma Humano , Humanos , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética
19.
J Am Chem Soc ; 132(21): 7231-3, 2010 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-20459096

RESUMO

Telomeric repeat-containing RNA is a new noncoding RNA molecule recently discovered in mammalian cells. Here we report the structural features of human telomere RNA r(UAGGGU) in the presence of K(+) and Na(+). We demonstrated for the first time that a novel U-tetrad is formed at the 3' end of a parallel human telomeric RNA G-quadruplex. The U-tetrad dramatically stabilizes human telomeric RNA G-quadruplex structure, leading to an increase in melting temperature (T(m)) of 29 degrees C. The U-tetrad-stabilized telomeric RNA G-quadruplex structure adds considerably to our understanding of the diversity of RNA G-quadruplex architectures. It shows that the structure of base "quartets" is important in RNA assembly. The structural information will be invaluable for understanding the function of human telomere RNA.


Assuntos
Quadruplex G , Estabilidade de RNA , RNA não Traduzido/química , Telômero/química , Humanos , Potássio/química , Sódio/química , Telômero/genética
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