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1.
Nucleic Acids Res ; 47(17): e102, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31318972

RESUMO

Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide-borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The 'TdT in rNTP-borate' method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems.


Assuntos
Boratos/química , DNA Nucleotidilexotransferase/metabolismo , Oligodesoxirribonucleotídeos/química , Ribonucleotídeos/química , Compostos Alílicos/química , Biotina/química , Tampões (Química) , DNA Nucleotidilexotransferase/antagonistas & inibidores , Oligodesoxirribonucleotídeos/biossíntese , Polimerização , Nucleosídeos de Purina/química
2.
Anal Bioanal Chem ; 411(22): 5779-5784, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31209546

RESUMO

Prostate-specific antigen (PSA) is the only biomarker for the diagnosis of prostate cancer. So the PSA screening test is very important due to the high occurrence of prostate cancer in men. In this work, a label-free fluorescent method was developed based on terminal deoxynucleotidyl transferase (TdT) and G-quadruplex-thioflavin T complex for detecting PSA. In the absence of PSA, the PSA aptamer can be used as the primer for TdT extension reactions, resulting in the formation of G-quadruplexes and generation of strong fluorescent signals. After the addition of PSA, the PSA-aptamer complex prevented the TdT extension reaction due to steric hindrance, thus resulting in a poor fluorescent signal. The assay showed a wide linear range (0.1 to 80 pg/mL) and a detection limit of 0.086 pg/mL (S/N = 3). It also has good specificity for PSA determination and gives satisfactory results when applied to biological samples. Conceivably, its merits such as good selectivity and high sensitivity indicate that the proposed method has a promising application potential in the clinical diagnosis and treatment of prostate cancer. Graphical abstract.


Assuntos
Benzotiazóis/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Antígeno Prostático Específico/sangue , Fluorescência , Humanos , Masculino
3.
Nat Commun ; 10(1): 2383, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160595

RESUMO

DNA is an emerging medium for digital data and its adoption can be accelerated by synthesis processes specialized for storage applications. Here, we describe a de novo enzymatic synthesis strategy designed for data storage which harnesses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) in kinetically controlled conditions. Information is stored in transitions between non-identical nucleotides of DNA strands. To produce strands representing user-defined content, nucleotide substrates are added iteratively, yielding short homopolymeric extensions whose lengths are controlled by apyrase-mediated substrate degradation. With this scheme, we synthesize DNA strands carrying 144 bits, including addressing, and demonstrate retrieval with streaming nanopore sequencing. We further devise a digital codec to reduce requirements for synthesis accuracy and sequencing coverage, and experimentally show robust data retrieval from imperfectly synthesized strands. This work provides distributive enzymatic synthesis and information-theoretic approaches to advance digital information storage in DNA.


Assuntos
Apirase/metabolismo , DNA Nucleotidilexotransferase/metabolismo , DNA/síntese química , Armazenamento e Recuperação da Informação/métodos , Nanoporos , Análise de Sequência de DNA
4.
Talanta ; 202: 152-158, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171163

RESUMO

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world, which can lead to considerably high mortality rate. It was reported that the prognosis is extremely poor and survival is often measured in months once CRC metastases become clinically evident. Therefore, the development of effective approach for metastatic CRC cells detection and imaging may potentially be significant and helpful for CRC prognosis and treatment. Therefore, we proposed a sensitive and specific approach for high-metastatic CRC LoVo cells detection and imaging by using terminal deoxynucleotidyl transferase (TdT)-initiated molecule beacons (MBs) arrayed fluorescent aptamer probes (denoted as TMAP). In this approach, the aptamer W3 targeting high-metastatic CRC LoVo cells was elongated to form W3-poly A at the 3'-hydroxyl terminus with repeated A bases in the presence of TdT and dATP. The MBs designed with poly T sequence in the loop were then hybridized with the poly A in the aptamer W3. The TMAP was easily constructed without the need of aptamer modification. It was demonstrated that this approach could specifically detect and image the high-metastatic CRC LoVo cells from the mixture of high-metastatic CRC LoVo cells and non-metastatic HCT-8 cells. Compared with 6-carboxyfluorescein (6-FAM) labeled aptamer W3, the TMAP was demonstrated to have a much stronger fluorescence signal on the target cells, realizing a 4-fold increase in signal-to-background ratio (SBR). Determination by flow cytometry allowed for detection of as low as 23 CRC LoVo cells in 200 µL cell culture medium. The high sensitivity and the capability for using in complicate biological samples imply that this approach holds considerable potential for metastatic CRC detection and therapy.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Neoplasias Colorretais/diagnóstico por imagem , DNA Nucleotidilexotransferase/química , Corantes Fluorescentes/química , Neoplasias Colorretais/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Espectrometria de Fluorescência , Células Tumorais Cultivadas
5.
Nucleic Acids Res ; 47(15): e85, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31114914

RESUMO

Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3'-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base-color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.


Assuntos
Mapeamento Cromossômico/métodos , DNA de Cadeia Simples/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sulfitos/química , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Metilação de DNA , DNA Nucleotidilexotransferase/genética , DNA Nucleotidilexotransferase/metabolismo , DNA de Cadeia Simples/metabolismo , Biblioteca Genômica , Humanos , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo
6.
Food Chem ; 294: 19-26, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126452

RESUMO

A novel aptasensor, based on a perylene probe (PAPDI; N,N'-bis(propylenetrimethylammonium)-3,4,9,10-perylenediimide), was developed for the detection of aflatoxin B1 (AFB1) in maize samples. AuNPs/DNA composites were synthesized and integrated with aptamers-modified magnetic nanoparticles (MNPs) via DNA hybridization. For AFB1 determination, AuNPs/DNA composites were released from MNPs through specific binding of AFB1 with the aptamer and used for assembly of the PAPDI probe. To enhance the method sensitivity, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA polymerization was performed to elongate DNA on AuNPs/DNA composites. As a result, more PAPDI probes were assembled on the AuNPs/DNA composites. Through a multiple signal amplification strategy, the proposed method exhibited high sensitivity towards AFB1, with a detection limit of 0.01 nM (3.1 pg/mL). In summary, the proposed method has great potential to be a universal strategy for monitoring AFB1 in food samples.


Assuntos
Aflatoxina B1/análise , DNA Nucleotidilexotransferase/metabolismo , DNA/química , Corantes Fluorescentes/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/metabolismo , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Nanocompostos/química , Hibridização de Ácido Nucleico , Zea mays/química , Zea mays/metabolismo
7.
Nucleic Acids Res ; 47(13): 6932-6945, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31001622

RESUMO

Acanthamoeba polyphaga mimivirus is an amoeba-infecting giant virus with over 1000 genes including several involved in DNA replication and repair. Here, we report the biochemical characterization of gene product 577 (gp577), a hypothetical protein (product of L537 gene) encoded by mimivirus. Sequence analysis and phylogeny suggested gp577 to be a primase-polymerase (PrimPol)-the first PrimPol to be identified in a nucleocytoplasmic large DNA virus (NCLDV). Recombinant gp577 protein purified as a homodimer and exhibited de novo RNA as well as DNA synthesis on circular and linear single-stranded DNA templates. Further, gp577 extends a DNA/RNA primer annealed to a DNA or RNA template using deoxyribonucleoties (dNTPs) or ribonucleotides (NTPs) demonstrating its DNA/RNA polymerase and reverse transcriptase activity. We also show that gp577 possesses terminal transferase activity and is capable of extending ssDNA and dsDNA with NTPs and dNTPs. Mutation of the conserved primase motif residues of gp577 resulted in the loss of primase, polymerase, reverse transcriptase and terminal transferase activities. Additionally, we show that gp577 possesses translesion synthesis (TLS) activity. Mimiviral gp577 represents the first protein from an NCLDV endowed with primase, polymerase, reverse transcriptase, terminal transferase and TLS activities.


Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mimiviridae/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/genética , DNA Nucleotidilexotransferase/isolamento & purificação , DNA Primase/química , DNA Primase/genética , DNA Primase/isolamento & purificação , Primers do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Dimerização , Mimiviridae/genética , RNA , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
8.
Nucleic Acids Res ; 47(7): 3272-3283, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30818397

RESUMO

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3' terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3' termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30-50 2'-deoxyribonucleotides to the 3' terminus of RNA molecules of various lengths and sequences, and extends RNA 3' termini with an assortment of 2'-deoxy and 2',3'-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3' terminal modification of RNA and potentially enzymatic synthesis of RNA.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/metabolismo , DNA Polimerase Dirigida por DNA/química , Humanos , RNA Mensageiro/genética
9.
Anal Biochem ; 567: 85-89, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30157446

RESUMO

Terminal deoxynucleotidyl transferase (TdT) is a unique template-free polymerase that randomly adds multiple deoxyribonucleoside triphosphates (dNTPs) to the 3'-OH terminus of ssDNA. This characteristic makes TdT a versatile enzymatic tool in many fields. Moreover, aberrant TdT expression is a well-recognized biomarker of several leukemic diseases and is related to carcinogenesis. In this study, we developed a facile, rapid, label-free, and convenient assay for TdT detection. TdT-generated poly A tails formed a fluorescent enhancement complex in the presence of coralyne. To achieve a better signal-to-noise ratio, we used potassium thiocyanate (KSCN), instead of other halogen anions (KCl, KBr, KI, NaI) as the quenching agent of dissociate coralyne. Our results demonstrate that this assay is extremely facile, rapid, and label-free; at levels as low as 0.025 U/mL, TdT was distinctly detected within 55 min. And the determination of TdT activity in RBL-2H3 and Reh cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis.


Assuntos
Adenosina/química , Alcaloides de Berberina/química , Técnicas Biossensoriais/métodos , DNA Nucleotidilexotransferase/análise , Polímeros/química , DNA Nucleotidilexotransferase/metabolismo , DNA de Cadeia Simples/química , Corantes Fluorescentes/química
10.
Cell Death Dis ; 9(9): 899, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30185775

RESUMO

Phospholipids are asymmetrically distributed across mammalian plasma membrane with phosphatidylserine (PS) and phosphatidylethanolamine concentrated in the cytoplasmic leaflet of the membrane bilayer. This asymmetric distribution is dependent on a group of P4-ATPases named PS flippases. The proper transport and function of PS flippases require a ß-subunit transmembrane protein 30 A (TMEM30A). Disruption of PS flippases led to several human diseases. However, the roles of TMEM30A in the central nervous system remain elusive. To investigate the role of Tmem30a in the cerebellum, we developed a Tmem30a Purkinje cell (PC)-specific knockout (KO) mouse model. The Tmem30a KO mice displayed early-onset ataxia and progressive PC death. Deficiency in Tmem30a led to an increased expression of Glial fibrillary acidic protein and astrogliosis in regions with PC loss. Elevated C/EBP homologous protein and BiP expression levels indicated the presence of endoplasmic reticulum stress in the PCs prior to visible cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis suggested that apoptotic cell death occurred in the cerebellum. Our data demonstrate that loss of Tmem30a in PCs results in protein folding and transport defects, a substantial decrease in dendritic spine density, increased astrogliosis and PC death. Taken together, our data demonstrate an essential role of Tmem30a in the cerebellum PCs.


Assuntos
Ataxia Cerebelar/metabolismo , Proteínas de Membrana/metabolismo , Células de Purkinje/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Apoptose/fisiologia , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Cerebelo/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos
12.
Biomacromolecules ; 19(8): 3525-3535, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-30011192

RESUMO

We synthesized long, nucleobase-modified, single-stranded DNA (ssDNA) using terminal deoxynucleotidyl transferase (TdT) enzymatic polymerization. Specifically, we investigated the effect of unnatural nucleobase size and incorporation density on ssDNA resistance to exo- and endonuclease degradation. We discovered that increasing the size and density of unnatural nucleobases enhances ssDNA resistance to degradation in the presence of exonuclease I, DNase I, and human serum. We also studied the mechanism of this resistance enhancement using molecular dynamics simulations. Our results show that the presence of unnatural nucleobases in ssDNA decreases local chain flexibility and hampers nuclease access to the ssDNA backbone, which hinders nuclease binding to ssDNA and slows its degradation. Our discoveries suggest that incorporating nucleobase-modified nucleotides into ssDNA, using enzymatic polymerization, is an easy and efficient strategy to prolong and tune the half-life of DNA-based materials in nucleases-containing environments.


Assuntos
DNA de Cadeia Simples/síntese química , Desoxirribonucleases/metabolismo , Biocatálise , DNA Nucleotidilexotransferase/metabolismo , DNA de Cadeia Simples/química , Hidrólise , Ligação Proteica , Nucleosídeos de Purina/química
13.
Mikrochim Acta ; 185(8): 380, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30027345

RESUMO

The article describes an on-chip amplification scheme initiated by a terminal deoxynucleotidyl transferase (TdT) for highly sensitive fluorometric determination of protein. Two thrombin-binding aptamers were designed to capture thrombin as they can form a sandwich structure for improved specificity. An amino-modified aptamer (TBA29) was first immobilized on a silicon chip. After capture of thrombin, a second aptamer (TBA15) was conjugated to the second binding site of thrombin. The 3'-terminal of aptamer TBA15 is exposed on the chip surface, and then fluorescein-labeled 12-dATP associates to the 3'-terminal with the help of TdT. This results in signal amplification, and eventually leads to highly sensitive detection. Under optimal conditions, fluorescence intensity is linearly related to the logarithm of thrombin concentration in the range of 100 fM - 0.1 µM, and the detection limit is as low as 2.0 fM. The assay is sensitive and selective even over potentially interfering proteins and in the presence of human serum. Graphical abstract Schematic strategy for thrombin detection. Two thrombin-binding aptamers were designed to capture thrombin to form a sandwich structure for improved specificity. The protein detection is based on TdT initiated on-chip fluorescent amplification.


Assuntos
Técnicas Biossensoriais/métodos , DNA Nucleotidilexotransferase/metabolismo , Fluorometria/métodos , Limite de Detecção , Trombina/análise , Aptâmeros de Nucleotídeos/metabolismo , Humanos , Silício/química , Trombina/metabolismo
14.
Luminescence ; 33(7): 1157-1163, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30047621

RESUMO

Here we report a novel bioluminescence (BL) method for exonuclease I (Exo I) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated pyrophosphate release. An inert hairpin probe with a blocked protruding 3'-terminal biotinylated nucleotide was designed, and a blocked 3'-terminal nucleotide of the probe would be removed only in the presence of Exo I, thus rendering the probe with a free 3'-hydroxyl group. Subsequently, with nucleotide incorporation by TdT, huge amounts of pyrophosphates were generated. After the conversion of pyrophosphate to adenosine triphosphate (ATP) through ATP sulfurylase, BL was emitted by the reaction of d-luciferin and ATP with firefly luciferase. Therefore, Exo I activity was indirectly quantified in the range 1-500 mU with a detection limit of 0.05 mU/µl. Moreover, the developed approach was successfully applied to investigate the inhibitory effect of streptavidin on cleavage of Exo I and also determine Exo I activity in spiked serum samples. Overall, the proposed method has high sensitivity and selectivity, and can be universally extended to the detection of other nucleases using terminal extension as a signal amplification method and BL as a detection signal, having potential application in the diagnosis of nuclease-related diseases or evaluation of nuclease functions in biological systems.


Assuntos
DNA Nucleotidilexotransferase/química , Enzimas Reparadoras do DNA/química , Difosfatos/química , Ensaios Enzimáticos/métodos , Exodesoxirribonucleases/química , Medições Luminescentes/métodos , Trifosfato de Adenosina/química , DNA Nucleotidilexotransferase/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Luciferina de Vaga-Lumes/química , Humanos , Limite de Detecção , Luminescência
15.
Anal Chem ; 90(14): 8629-8634, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29911858

RESUMO

As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3'-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.


Assuntos
DNA Nucleotidilexotransferase/metabolismo , Ensaios Enzimáticos/métodos , Exodesoxirribonucleases/metabolismo , Uracila-DNA Glicosidase/análise , Técnicas Biossensoriais/métodos , Células HeLa , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico/métodos , Uracila-DNA Glicosidase/metabolismo
16.
BMJ Case Rep ; 20182018 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884716

RESUMO

Terminal deoxynucleotidyl transferase (TdT)-negative T-cell lymphoblastic lymphoma is a variant of T-cell lymphoblastic lymphoma/T-cell lymphoblastic leukaemia. TdT is a marker of immaturity expressed in 90%-95% cases of lymphoblastic lymphoma and useful in differentiating it from other mature lymphomas/leukaemias. It has been associated with poorer response to chemotherapy and a more aggressive outcome. Here we present a case of TdT-negative T-cell lymphoblastic lymphoma in a 28-year-old man who presented with superior vena cava syndrome. The patient was treated with hyper-cyclophosphamide,vincristine, Adriamycin, dexamethasone (CVAD), however unfortunately suffered a relapse 1 year later. A unique feature of our case was that on relapse, the patient lost expression of the T-cell lineage-specific marker CD3, which has previously not been reported in association with TdT-negative T-cell lymphoblastic lymphoma. The patient failed to respond to chemotherapy on his relapse and died.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Complexo CD3/deficiência , DNA Nucleotidilexotransferase/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adulto , Biomarcadores/metabolismo , Evolução Fatal , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Recidiva
17.
Mikrochim Acta ; 185(5): 280, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29725866

RESUMO

A method is reported for the fluorometric quantitation of microRNA. It is making use of a luminescent probe deribed from terbium(III) ion whose fluorescence is sensitized with a guanine-rich (G-rich) nucleotide. The probe has a large Stokes' shift and strong and sharp emission bands. The assay relies on the wide substrate specificity of terminal deoxynucleotidyl transferase (TdTase), which catalyzes the formation of long G-rich nucleotides when using microRNA primer as a trigger to start the polymerization. The addition of Tb(III) induces the formation of a G-quadruplex from the G-rich nucleotide, and this strongly enhances the green fluorescence of Tb(III) (peaking at 545 nm upon photoexcitation at 290 nm). Specifically, microRNA-21 was chosen as the analyte. The fluorescence intensity of Tb(III) increases linearly in the 1 pM to 1 nM microRNA concentration range, and the detection limit is as low as 0.11 pM. The method can distinguish between family members of microRNA and performs excellently even when applied to extracts of cancer cells. Graphical abstract A fluorometric technique is reported for the determination of microRNA. It is based on signal enhancement based on the sensitization of terbium(III) via a guanine-rich nucleotide sequence. Klenow Fragment exo- (KFexo-) generates DNA sequence at the 3'-OH of microRNA, and terminal deoxynucleotidyl transferase (TdTase) catalyzes the formation of long G-rich nucleotides.


Assuntos
Técnicas Biossensoriais/métodos , DNA Nucleotidilexotransferase/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Medições Luminescentes/métodos , MicroRNAs/análise , Térbio/química , Células A549 , Humanos , Células MCF-7
18.
Nucleic Acids Res ; 46(16): e95, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-29846671

RESUMO

Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3'-azide-modified dideoxyribonucleotide onto the 3'-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3'-end to a 5'-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.


Assuntos
DNA de Cadeia Simples , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Triazóis/química , Alquinos/química , Azidas/química , Química Click , Cobre/química , Reação de Cicloadição , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/metabolismo , Nuclease do Micrococo/genética , Nuclease do Micrococo/metabolismo , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA/métodos
19.
Curr Opin Struct Biol ; 53: 22-31, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29656238

RESUMO

Terminal deoxynucleotidyltransferase (TdT) is a member of the polX family which is involved in DNA repair. It has been known for years as an untemplated DNA polymerase used during V(D)J recombination to generate diversity at the CDR3 region of immunoglobulins and T-cell receptors. Recently, however, TdT was crystallized in the presence of a complete DNA synapsis made of two double-stranded DNA (dsDNA), each with a 3' protruding end, and overlapping with only one micro-homology base-pair, thus giving structural insight for the first time into DNA synthesis across strands. It was subsequently shown that TdT indeed has an in trans template-dependent activity in the presence of an excess of the downstream DNA duplex. A possible biological role of this dual activity is discussed.


Assuntos
DNA Nucleotidilexotransferase , Animais , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidilexotransferase/fisiologia , Reparo do DNA , Enzimas Reparadoras do DNA/fisiologia , DNA Polimerase Dirigida por DNA/química , Humanos , Recombinação V(D)J
20.
Anal Chem ; 90(8): 5390-5397, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29600844

RESUMO

A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N3, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N3-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3'-OH termini exposed outside. Then terminal deoxynucleotidyl transferase (TdT), a sequence-independent and template-free polymerase, would specifically catalyze the DNA polymerization along these 3'-OH termini on the MBs, forming poly(T) tails up to thousands of nucleotides long. Each poly(T) tail allows specific binding of numerous 6-carboxyfluorescein (FAM)-labeled poly(A)25 oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. By integrating the advantages of CCNAL-TEP for highly efficient signal amplification and robust MBs signal readout with powerful flow cytometer, high sensitivity is achieved and the detection limit of plant miRNA has been pushed down to a low level of 5 fM with high specificity to well discriminate even single-base difference between miRNA targets.


Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA/metabolismo , Citometria de Fluxo , MicroRNAs/análise , Arabidopsis/química , Química Click , DNA/química , Metilação , Polimerização
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