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1.
Talanta ; 204: 29-35, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357296

RESUMO

Because STAT3 is a potent proto-oncogene, screening STAT3 gene has potential for use in tumor diagnosis, classification of subtypes, and molecular target therapy. Thus, in this study, using STAT3 gene as the model molecule, we developed a novel amplification strategy, ultrasensitive rolling circle amplification (THP-RCA) based on target-catalyzed hairpin structure-mediated padlock cyclization, for the ultrasensitive detection of human proto-oncogenes in a homogenous solution. In this system, HP1 was designed as the cyclization template and RCA reaction primer, while HP2 was the padlock probe. The two probes can fold into a hairpin structure via the self-hybridization and thus lock the signaling process in the absence of target species. The hybridization of HP2 with HP1 in an end-to-end fashion occurs with the help of target DNA. Subsequently, HP2 is cyclized by ligase on HP1 template. Interestingly, during the hybridization and enzymatic cyclization of HP2, the target DNA only serves as the catalytic probe and is not exhausted. The cyclized HP2 enables the rolling circle amplification, generating a long tandem single-stranded (ss) DNA product that is capable of hybridizing with considerable quantity of molecular beacons (MBs). As a result, the dramatically amplified fluorescence value is achieved for the ultrasensitive detection of the STAT3 gene. As a result, target DNA is able to be quantified down to 100 fM with a high specificity towards wild-type target DNA. Moreover, the sensing system is suitable for the target detection in human serum. The novel sensing strategy shows tremendous prospect for application in tumor diagnosis and clinical therapy guidance.


Assuntos
Técnicas Biossensoriais/métodos , DNA/sangue , DNA/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Fator de Transcrição STAT3/genética , Fagos Bacilares/enzimologia , Bacteriófago T4/enzimologia , Ciclização , DNA/genética , DNA Polimerase Dirigida por DNA/química , Corantes Fluorescentes/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico , Compostos Orgânicos/química , Espectrometria de Fluorescência/métodos , Proteínas Virais/química
2.
Analyst ; 144(16): 4795-4802, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31274133

RESUMO

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP). The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29; thus, amplified detection of the target can be achieved. To our knowledge, this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria. It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3' → 5' proofreading exonuclease activity of phi29, which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension. On the basis of the present amplification strategy, our biosensor exhibits excellent specificity and sensitivity toward S. typhimurium with an excellent detection limit as low as 1.5 cfu mL-1. In addition, the strategy offers the advantages of a simplified operation, shortened analysis time, and highly sensitive detection of pathogens with only a one-step reaction. Furthermore, by redesigning the corresponding binding molecules, the proposed strategy can be easily extended for the detection of a wide spectrum of analytes. Hence, the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.


Assuntos
DNA Bacteriano/análise , DNA Polimerase Dirigida por DNA/química , Salmonella typhimurium/isolamento & purificação , Fagos Bacilares/enzimologia , Bacillus subtilis , Técnicas Biossensoriais/métodos , Sondas de DNA/química , Sondas de DNA/genética , DNA Bacteriano/genética , Escherichia coli , Fluoresceínas/química , Corantes Fluorescentes/química , Sequências Repetidas Invertidas , Limite de Detecção , Listeria , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência , Proteínas Virais/química
3.
Mikrochim Acta ; 186(6): 344, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076917

RESUMO

A method is described for counting circulating tumor cells (CTCs). It is making use of inductively coupled plasma mass spectrometry (ICP-MS) along with a dual amplification strategy by combining rolling circle amplification (RCA) and gold nanoparticle (Au NP) labeling. HepG2 cells, as a representative CTC line, were captured by anti-epithelial cellular adhesion molecule (EpCAM) immobilized on a microplate, then specifically labeled with biotinylated anti-asialoglycoprotein receptor (ASGPR). Taking streptavidin (SA) as the bridge, the biotinylated RCA primer was conjugated to HepG2 cells. When the RCA reaction was triggered, long ssDNA with tandem repeats generated on the cell surface. Then, Au NP functionalized detection DNA (signal probes) was added to hybridize with the ssDNA. After removing the redundant signal probes, Au NPs conjugated on target HepG2 cells were subjected to ICP-MS detection. By adopting such a dual amplification strategy, a 756-fold improvement in sensitivity is accomplished compared to the method involving only Au NP labeling without RCA. The limit of detection is as low as 3 HepG2 cells (15 cell mL-1) which is the lowest LOD in ICP-MS based methods for cell counting. Besides, the method provides good selectivity, a wide linear range of 10-1000 HepG2 cells (50-5000 cells mL-1), and relative standard deviations of 6.3% (n = 7; 50 HepG2 cells (250 cells mL-1)). The method was successfully applied to HepG2 cell counting in spiked human blood samples and gave good recoveries. Graphical abstract Schematic presentation of an ICP-MS based immunoassay for the sensitive circulating tumor cells counting by combining rolling circle amplification (RCA) with gold nanoparticle (Au NP) labeling. ICP-MS: inductively coupled plasma mass spectrometry; ASGPR: asialoglycoprotein receptor; EpCAM: epithelial cellular adhesion molecule.


Assuntos
Contagem de Células/métodos , Nanopartículas Metálicas/química , Células Neoplásicas Circulantes/química , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Fagos Bacilares/enzimologia , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , DNA Polimerase Dirigida por DNA/química , Molécula de Adesão da Célula Epitelial/imunologia , Ouro/análise , Ouro/química , Células Hep G2 , Humanos , Imunoensaio/métodos , Espectrometria de Massas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Proteínas Virais/química
4.
Analyst ; 144(10): 3389-3397, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-30990481

RESUMO

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Nanoestruturas/química , Ocratoxinas/análise , Aptâmeros de Nucleotídeos/genética , Fagos Bacilares/enzimologia , Bacteriófago T4/enzimologia , Sequência de Bases , DNA/genética , DNA Ligases/química , DNA Polimerase Dirigida por DNA/química , Desoxirribonuclease IV (Fago T4-Induzido)/química , Fluorescência , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Quadruplex G , Sequências Repetidas Invertidas , Limite de Detecção , Mesoporfirinas/química , Técnicas de Amplificação de Ácido Nucleico , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ocratoxinas/química , Espectrometria de Fluorescência/métodos , Proteínas Virais/química , Vinho/análise
5.
Appl Environ Microbiol ; 85(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552194

RESUMO

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.


Assuntos
Fagos Bacilares/enzimologia , Bacillus cereus/virologia , Domínio Catalítico , Endopeptidases/metabolismo , Esporos Bacterianos/virologia , Anti-Infecciosos , Fagos Bacilares/genética , Fagos Bacilares/isolamento & purificação , Bacillus cereus/metabolismo , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Especificidade de Hospedeiro , Modelos Moleculares , Peptidoglicano/metabolismo , Mutação Puntual , Conformação Proteica , Domínios Proteicos/genética , Alinhamento de Sequência , Esporos Bacterianos/metabolismo
6.
J Am Chem Soc ; 140(51): 18093-18103, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30427676

RESUMO

The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small-molecule adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small-molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.


Assuntos
Evolução Molecular Direcionada/métodos , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Fagos Bacilares/enzimologia , DNA Polimerase Dirigida por DNA/genética , Doxorrubicina/farmacologia , Resistência a Medicamentos/genética , Células HEK293 , Humanos , Integrases/genética , Leucina-tRNA Ligase/genética , Mutagênese , Peptídeo Hidrolases/genética , Estudo de Prova de Conceito , Engenharia de Proteínas , Fatores de Transcrição/genética , Proteínas Virais/genética
7.
Nucleic Acids Res ; 46(15): 7495-7505, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30010979

RESUMO

Recently reported DNA nanoflowers are an interesting class of organic-inorganic hybrid materials which are prepared using DNA polymerases. DNA nanoflowers combine the high surface area and scaffolding of inorganic Mg2P2O7 nanocrystals with the targeting properties of DNA, whilst adding enzymatic stability and enhanced cellular uptake. We have investigated conditions for chemically modifying the inorganic core of these nanoflowers through substitution of Mg2+ with Mn2+, Co2+ or Zn2+ and have characterized the resulting particles. These have a range of novel nanoarchitectures, retain the enzymatic stability of their magnesium counterparts and the Co2+ and Mn2+ DNA nanoflowers have added magnetic properties. We investigate conditions to control different morphologies, DNA content, hybridization properties, and size. Additionally, we show that DNA nanoflower production is not limited to Ф29 DNA polymerase and that the choice of polymerase can influence the DNA length within the constructs. We anticipate that the added control of structure, size and chemistry will enhance future applications.


Assuntos
Cobalto/química , DNA Polimerase Dirigida por DNA/química , DNA/síntese química , Manganês/química , Nanopartículas Metálicas/química , Oligonucleotídeos/síntese química , Zinco/química , Fagos Bacilares/enzimologia , Nanotecnologia/métodos
8.
Proc Natl Acad Sci U S A ; 115(31): 7961-7966, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30012596

RESUMO

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.


Assuntos
Adenosina Trifosfatases , Fagos Bacilares/enzimologia , Modelos Biológicos , Proteínas Virais , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
9.
Langmuir ; 34(49): 14882-14890, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30044093

RESUMO

Single-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. Because ssDNA is more flexible than double-stranded (ds) DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate that low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription, and repair.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteína de Replicação A/metabolismo , Fagos Bacilares/enzimologia , Sequência de Bases , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Fluorescência , Proteínas de Fluorescência Verde/química , Humanos , Conformação de Ácido Nucleico/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Proteína de Replicação A/química , Cloreto de Sódio/química
10.
Viruses ; 10(5)2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29883383

RESUMO

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0⁻8.0), over a broad temperature range (4⁻55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.


Assuntos
Fagos Bacilares/enzimologia , Bacillus/virologia , Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Fagos Bacilares/classificação , Fagos Bacilares/genética , Domínio Catalítico , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/farmacologia , Estabilidade Enzimática , Especificidade de Hospedeiro , Modelos Moleculares , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/farmacologia
11.
Microb Pathog ; 119: 221-224, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29678741

RESUMO

Drug-resistant Gram-positive pathogens have been a rising risk in hospitals and food industries from the last decades. Here in, the potential of endolysin production in Dasht Desert Bacterial Culture Collection (DDBCC), against indicator bacteria, was investigated. DDBCC was screened against autoclaved-indicator bacteria; Streptococcus faecalis, Streptococcus pyogenes, Bacillus sp, Bacillus subtilis and Staphylococcus aureus as the substrates for the endolysin enzymes. The endolysins were produced in BHI medium followed by ammonium sulfate purification. Peptidoglycan hydrolytic activity was tested by zymogram method. Lysogenic bacteria were induced by 0.1 µg/ml mitomycin C for bacteriophages extraction. The lysogenic bacteria inhibited S. pyogenes, S. faecalis, Bacillus sp. and B. subtilis. The strain DDBCC10 was selected for further experiments on its higher and specific activity against the cell wall of S. faecalis. The highest activity for the endolysin was obtained at 50-60% ammonium sulfate saturation as 8 U/ml. Lys10, a 22 kDa enzyme, digested the cell wall of S. faecalis in 15 min while the whole phage from DDBCC10 could form plaque on S. faecalis and S. pyogenes. In a Transmission Electron Microscopy assay (TEM), the phage was distinguished as a member of Siphoviridae. Here; Lys10 is introduced as a new biocontrol agent against S. faecalis for therapeutics, disinfection, and food preservatives purposes at a much lower expense than recombinant endolysins.


Assuntos
Antibacterianos/farmacologia , Fagos Bacilares/enzimologia , Bacillus subtilis/virologia , Endopeptidases/farmacologia , Fagos Bacilares/isolamento & purificação , Bactérias/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Endopeptidases/química , Endopeptidases/isolamento & purificação , Ensaio de Placa Viral
12.
Nucleic Acids Res ; 46(7): 3625-3632, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29554297

RESUMO

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA/química , DNA Polimerase Dirigida por RNA/química , RNA/química , Fagos Bacilares/enzimologia , Sequência de Bases , DNA/genética , DNA Circular , DNA Polimerase Dirigida por DNA/genética , RNA/genética , DNA Polimerase Dirigida por RNA/genética , Análise de Sequência de DNA
13.
Proc Natl Acad Sci U S A ; 115(13): E2921-E2929, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531047

RESUMO

Most replicative DNA polymerases (DNAPs) are endowed with a 3'-5' exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp121 of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp90 in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.


Assuntos
Ácido Aspártico/genética , Fagos Bacilares/enzimologia , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação , Sequência de Aminoácidos , Fagos Bacilares/genética , DNA Polimerase Dirigida por DNA/genética , Exodesoxirribonucleases/genética , Mutagênese Sítio-Dirigida , Homologia de Sequência
14.
Chem Commun (Camb) ; 54(17): 2158-2161, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29431761

RESUMO

The isothermal amplification of DNA in minimally buffered conditions allows to perform and monitor nucleic acid amplification with minimal technological and operative requirements. We show in this work how phi29 can operate multiple displacement amplification in minimally buffered conditions producing, as a readout, pH shifts attaining subunits of pH.


Assuntos
Fagos Bacilares/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Tampões (Química) , DNA Viral/genética , Genoma Viral , Papillomavirus Humano 16/genética , Humanos , Concentração de Íons de Hidrogênio
15.
Anal Chem ; 90(1): 1029-1034, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29210271

RESUMO

Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer. Herein, we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells (human Burkitt's lymphoma cells), by combining the enzymatic signal amplification with an aerolysin nanopore sensor. In this assay, an aptamer for Ramos cells was prehybridized with a short complementary DNA. The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA, which would subsequently trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce characteristic current events when translocated through aerolysin. The proposed method exhibits excellent sensitivity, and as few as 5 Ramos cells could be detected. With good selectivity, the approach can allow for the determination of cancer cells in human serum, offering a powerful tool for biomedical research and clinical diagnosis.


Assuntos
Toxinas Bacterianas/química , Bioensaio/métodos , Linfoma de Burkitt/diagnóstico , Nanoporos , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas Citotóxicas Formadoras de Poros/química , Aptâmeros de Nucleotídeos/genética , Fagos Bacilares/enzimologia , Brevibacillus/enzimologia , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA Polimerase Dirigida por DNA/química , Endodesoxirribonucleases/química , Humanos , Hibridização de Ácido Nucleico
16.
Nucleic Acids Res ; 45(10): 5958-5967, 2017 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-28402520

RESUMO

AR9 is a giant Bacillus subtilis phage whose uracil-containing double-stranded DNA genome encodes distant homologs of ß and ß' subunits of bacterial RNA polymerase (RNAP). The products of these genes are thought to assemble into two non-canonical multisubunit RNAPs - a virion RNAP (vRNAP) that is injected into the host along with phage DNA to transcribe early phage genes, and a non-virion RNAP (nvRNAP), which is synthesized during the infection and transcribes late phage genes. We purified the AR9 nvRNAP from infected B. subtilis cells and characterized its transcription activity in vitro. The AR9 nvRNAP requires uracils rather than thymines at specific conserved positions of late viral promoters. Uniquely, the nvRNAP recognizes the template strand of its promoters and is capable of specific initiation of transcription from both double- and single-stranded DNA. While the AR9 nvRNAP does not contain homologs of bacterial RNAP α subunits, it contains, in addition to the ß and ß'-like subunits, a phage protein gp226. The AR9 nvRNAP lacking gp226 is catalytically active but unable to bind to promoter DNA. Thus, gp226 is required for promoter recognition by the AR9 nvRNAP and may represent a new group of transcription initiation factors.


Assuntos
Fagos Bacilares/enzimologia , DNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Virais/metabolismo , Bacillus subtilis/virologia , Sítios de Ligação , Sequência Consenso , Pegada de DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Genes Virais , Multimerização Proteica , Subunidades Proteicas , Especificidade por Substrato , Moldes Genéticos , Transcrição Genética , Uracila/química , Proteínas Virais/isolamento & purificação
17.
mBio ; 8(1)2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28196958

RESUMO

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases.IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages.


Assuntos
Fagos Bacilares/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/virologia , RNA Polimerases Dirigidas por DNA/genética , Antibióticos Antituberculose/farmacologia , Fagos Bacilares/enzimologia , Fagos Bacilares/genética , Bacillus subtilis/efeitos dos fármacos , DNA Viral , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Interações Hospedeiro-Patógeno/genética , Regiões Promotoras Genéticas , Rifampina/farmacologia , Alinhamento de Sequência , Transcrição Genética , Proteínas Virais/metabolismo
18.
Cell ; 168(1-2): 186-199.e12, 2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-28041851

RESUMO

Bacteriophages (phages) typically exhibit a narrow host range, yet they tremendously impact horizontal gene transfer (HGT). Here, we investigate phage dynamics in communities harboring phage-resistant (R) and sensitive (S) bacteria, a common scenario in nature. Using Bacillus subtilis and its lytic phage SPP1, we demonstrate that R cells, lacking SPP1 receptor, can be lysed by SPP1 when co-cultured with S cells. This unanticipated lysis was triggered in part by phage lytic enzymes released from nearby infected cells. Strikingly, we discovered that occasionally phages can invade R cells, a phenomenon we termed acquisition of sensitivity (ASEN). We found that ASEN is mediated by R cells transiently gaining phage attachment molecules from neighboring S cells and provide evidence that this molecular exchange is driven by membrane vesicles. Exchange of phage attachment molecules could even occur in an interspecies fashion, enabling phage adsorption to non-host species, providing an unexplored route for HGT. VIDEO ABSTRACT.


Assuntos
Fagos Bacilares/fisiologia , Bacillus subtilis/virologia , Bacteriólise , Receptores Virais/metabolismo , Bacillus/virologia , Fagos Bacilares/enzimologia , Bacillus subtilis/metabolismo , Especificidade de Hospedeiro , Staphylococcus aureus/virologia , Transdução Genética
19.
Biosci Biotechnol Biochem ; 81(1): 135-146, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27885938

RESUMO

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.


Assuntos
Fagos Bacilares/genética , Fagos Bacilares/fisiologia , Bacillus subtilis/virologia , Genômica , Glicosídeo Hidrolases/genética , Ácido Poliglutâmico/análogos & derivados , Alimentos de Soja , Sequência de Aminoácidos , Fagos Bacilares/enzimologia , Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Cápsulas , Fermentação , Regulação Viral da Expressão Gênica , Genoma Viral/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Ácido Poliglutâmico/metabolismo , Solubilidade
20.
Protein Eng Des Sel ; 29(12): 617-628, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27672049

RESUMO

Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification. To be able to select for mezophilic enzymes we have employed multiple freezing-thawing cycles of emulsion as a bacterial cell wall disruption step instead of high temperature incubation. Subsequently WGA like plasmid DNA amplification could be performed by phi29 DNA polymerase applying different selection pressure conditions (temperature, buffer composition, modified dNTP, time, etc.). In our case the library of random phi29 DNA polymerase mutants was subjected to seven selection rounds of isothermal CSR (iCSR). After the selection polymerase variant containing the most frequent mutations was constructed and characterized. The mutant phi29 DNA polymerase can perform WGA at elevated temperatures (40-42°C), generate two to five times more of DNA amplification products, and has significantly increased half-life at 30 and 40°C, both in the presence or the absence of DNA substrate.


Assuntos
Fagos Bacilares/enzimologia , DNA Polimerase Dirigida por DNA/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Temperatura , Sequência de Aminoácidos , Fagos Bacilares/genética , DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Genômica , Meia-Vida , Mutação
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