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1.
Nat Commun ; 11(1): 4714, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948754

RESUMO

The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.


Assuntos
DNA/química , Fenômenos Magnéticos , Magnetismo/métodos , Nanotecnologia , Análise Espectral/métodos , Ciprofloxacino/farmacologia , DNA/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Conformação de Ácido Nucleico , Pinças Ópticas , Fenômenos Físicos , Software
2.
Nat Commun ; 11(1): 4784, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963245

RESUMO

Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase µ (Polµ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polµ engaging a DSB substrate. Synapsis is mediated solely by Polµ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol µ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polµ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/química , DNA/química , Cristalografia por Raios X , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica
3.
PLoS One ; 15(8): e0238322, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866178

RESUMO

Space-filling curves have been used for decades to study the folding principles of globular proteins, compact polymers, and chromatin. Formally, space-filling curves trace a single circuit through a set of points (x,y,z); informally, they correspond to a polymer melt. Although not quite a melt, the folding principles of Human chromatin are likened to the Hilbert curve: a type of space-filling curve. Hilbert-like curves in general make biologically compelling models of chromatin; in particular, they lack knots which facilitates chromatin folding, unfolding, and easy access to genes. Knot complexity has been intensely studied with the aid of Alexander polynomials; however, the approach does not generalize well to cases of more than one chromosome. Crossing complexity is an understudied alternative better suited for quantifying entanglement between chromosomes. Do Hilbert-like configurations limit crossing complexity between chromosomes? How does crossing complexity for Hilbert-like configurations compare to equilibrium configurations? To address these questions, we extend the Mansfield algorithm to enable sampling of Hilbert-like space filling curves on a simple cubic lattice. We use the extended algorithm to generate equilibrium, intermediate, and Hilbert-like configurational ensembles and compute crossing complexity between curves (chromosomes) in each configurational snapshot. Our main results are twofold: (a) Hilbert-like configurations limit entanglement between chromosomes and (b) Hilbert-like configurations do not limit entanglement in a model of S-phase DNA. Our second result is particularly surprising yet easily rationalized with a geometric argument. We explore ergodicity of the extended algorithm and discuss our results in the context of more sophisticated models of chromatin.


Assuntos
DNA/química , DNA/genética , Fase S/genética , Algoritmos , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Humanos , Polímeros/química
4.
Nature ; 586(7827): 151-155, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968275

RESUMO

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.


Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/metabolismo , Animais , Biocatálise , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Metilação de DNA , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética
5.
Nat Commun ; 11(1): 4437, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895378

RESUMO

Efficient search for DNA damage embedded in vast expanses of the DNA genome presents one of the greatest challenges to DNA repair enzymes. We report here crystal structures of human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, that interact with the DNA containing the damaged base oxoG and the normal base G while they are nested in the DNA helical stack. The structures reveal that hOGG1 engages the DNA using different protein-DNA contacts from those observed in the previously determined lesion recognition complex and other hOGG1-DNA complexes. By applying molecular dynamics simulations, we have determined the pathways taken by the lesion and normal bases when extruded from the DNA helix and their associated free energy profiles. These results reveal how the human oxoG DNA glycosylase hOGG1 locates the lesions inside the DNA helix and facilitates their extrusion for repair.


Assuntos
DNA Glicosilases/química , Reparo do DNA , Simulação de Dinâmica Molecular , Cristalografia por Raios X , DNA/química , Dano ao DNA , Conformação Proteica
6.
Nat Commun ; 11(1): 4826, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958757

RESUMO

DNA replication initiates from multiple genomic locations called replication origins. In metazoa, DNA sequence elements involved in origin specification remain elusive. Here, we examine pluripotent, primary, differentiating, and immortalized human cells, and demonstrate that a class of origins, termed core origins, is shared by different cell types and host ~80% of all DNA replication initiation events in any cell population. We detect a shared G-rich DNA sequence signature that coincides with most core origins in both human and mouse genomes. Transcription and G-rich elements can independently associate with replication origin activity. Computational algorithms show that core origins can be predicted, based solely on DNA sequence patterns but not on consensus motifs. Our results demonstrate that, despite an attributed stochasticity, core origins are chosen from a limited pool of genomic regions. Immortalization through oncogenic gene expression, but not normal cellular differentiation, results in increased stochastic firing from heterochromatin and decreased origin density at TAD borders.


Assuntos
DNA/biossíntese , DNA/química , Origem de Replicação/genética , Animais , Composição de Bases , Sequência de Bases , Carcinogênese , Diferenciação Celular , Células Cultivadas , Replicação do DNA/genética , Genoma Humano/genética , Heterocromatina/genética , Humanos , Camundongos , Motivos de Nucleotídeos , Transcrição Genética
7.
Nat Commun ; 11(1): 4747, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958761

RESUMO

Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical '30 nm' structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre-fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.


Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica
8.
Nat Commun ; 11(1): 4384, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873796

RESUMO

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , DNA/química , Nanoestruturas/química , Imagem Individual de Molécula/instrumentação , Biomarcadores/análise , Proteína C-Reativa/análise , Desenho de Equipamento , Humanos , Limite de Detecção , Nanotecnologia/métodos
9.
Phys Rev Lett ; 125(4): 048104, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32794805

RESUMO

The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.


Assuntos
Ecossistema , RNA/química , RNA/genética , Catálise , DNA/química , DNA/genética , DNA/metabolismo , Replicação do DNA , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Planeta Terra , Evolução Molecular , Temperatura Alta , Biossíntese de Proteínas/genética , RNA/metabolismo
10.
Science ; 369(6503): 566-571, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732424

RESUMO

CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.


Assuntos
Adenina/química , Adenosina Desaminase/química , Proteína 9 Associada à CRISPR/química , Sistemas CRISPR-Cas , DNA/química , Proteínas de Escherichia coli/química , Edição de Genes , Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , Microscopia Crioeletrônica , Desaminação , Proteínas de Escherichia coli/genética
11.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826907

RESUMO

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Assuntos
Proteínas de Bactérias/química , DNA Primase/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
12.
Nat Commun ; 11(1): 4263, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848132

RESUMO

Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação , Domínio AAA , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Hidrólise , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Free Radic Res ; 54(7): 517-524, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32781874

RESUMO

The present study reports radiation-chemical yields of 2.5-diaminoimidazolone (Iz) derivatives in X-irradiated phosphate-buffered solutions of guanosine and double-stranded DNA. Various gassing conditions (air, N20/O2 (4:1), N2O, vacuum) were employed to elucidate the contribution of several alternative pathways leading to Iz in reactions initiated by hydroxyl radical attack on guanine. In all systems, Iz was identified as the second by abundance guanine degradation product after 8-oxoguanine, formed in 1:5 (guanosine) and 1:3.3 (DNA) ratio to the latter in air-saturated solutions. Experimental data strongly suggest that the addition of molecular oxygen to the neutral guanine radical G(-H)• plays a major in Iz production in oxygenated solutions of double-stranded DNA while in other systems it may compete with recombination of G(-H)• with superoxide and/or alkyl peroxyl radicals. The production of Iz through hydroxyl radical attack on 8-oxoguanine was also shown to take place although the chemical yield of Iz (ca 6%) in this process is too low to compete with the other pathways. The linearity of Iz accumulation with dose also indicates a negligible contribution of this channel to its yield in all systems.


Assuntos
Dano ao DNA , DNA/química , Radicais Livres/química , Radical Hidroxila/química , Imidazóis/química , 8-Hidroxi-2'-Desoxiguanosina/química , Animais , DNA/efeitos da radiação , Diaminas/química , Guanosina/química , Masculino , Salmão
14.
Nat Commun ; 11(1): 4339, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859909

RESUMO

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.


Assuntos
Técnicas Citológicas/métodos , DNA/química , Microscopia de Fluorescência/métodos , Simulação de Acoplamento Molecular/métodos , Linhagem Celular , Humanos , Processamento de Imagem Assistida por Computador/métodos , Oligonucleotídeos , Coloração e Rotulagem/métodos
15.
PLoS One ; 15(8): e0236709, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790736

RESUMO

BACKGROUND: With the development of second-generation sequencing technology, more and more DNA sequence variations have been detected. Exon sequencing is the first choice for sequencing many cancer genes, and it can be better used to identify disease status by detecting gene variants. PCR sequence is an effective method to capture that sequence of an exon in the process of sequencing. Exon sequencing sequence contains PCR primer sequence, the correct position of the sequence can be determined by PCR primer sequence, which can be found in SNP, Indel mutation point by comparing the sequence of PCR primer sequence. RESULTS: In this paper, a matching algorithm based on the PCR primer sequence is proposed, which can effectively sequence the position of PCR primer sequence and find out the key position sequence. Then the sequencing sequence is sorted and the number of the same sequence is counted to reduce the matching times. Then, the sequenced sequence was matched with PCR primer sequence, so that the DNA position could be accurately matched and the variation in the sequenced sequence could be found more quickly. CONCLUSIONS: Compared with the traditional sequence matching method, PCR primer sequence matching method can match many sequences and find more variation. It also showed a high recall rate in the recall rate.


Assuntos
Algoritmos , Reação em Cadeia da Polimerase/métodos , DNA/química , DNA/genética , DNA/metabolismo , Primers do DNA/metabolismo , Éxons , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
16.
Lab Chip ; 20(19): 3560-3568, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32844858

RESUMO

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s-1 and maximum cooling rate of 8 °C s-1. Target DNA concentrations in the range from 101 ± 1.4 copies per µL to 260 000 ± 20 000 copies per µL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 µm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 µm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per µL to 25 889 ± 737 copies per µL.


Assuntos
Reação em Cadeia da Polimerase/instrumentação , Algoritmos , Técnicas Biossensoriais , DNA/química , Fontes de Energia Elétrica , Humanos , Membranas Artificiais , Miniaturização , Polimetil Metacrilato
17.
PLoS One ; 15(8): e0237356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817618

RESUMO

DESS is a formulation widely used to preserve DNA in biological tissue samples. Although it contains three ingredients, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA) and sodium chloride (NaCl), it is frequently referred to as a DMSO-based preservative. The effectiveness of DESS has been confirmed for a variety of taxa and tissues, however, to our knowledge, the contributions of each component of DESS to DNA preservation have not been evaluated. To address this question, we stored tissues of three aquatic taxa, Mytilus edulis (blue mussel), Faxonius virilis (virile crayfish) and Alitta virens (clam worm) in DESS, each component of DESS individually and solutions containing all combinations of two components of DESS. After storage at room temperature for intervals ranging from one day to six months, we extracted DNA from each tissue and measured the percentage of high molecular weight (HMW) DNA recovered (%R) and normalized HMW DNA yield (nY). Here, HMW DNA is defined as fragments >10 kb. For comparison, we also measured the %R and nY of HMW DNA from extracts of fresh tissues and those stored in 95% EtOH over the same time intervals. We found that in cases where DESS performed most effectively (yielding ≥ 20%R of HMW DNA), all solutions containing EDTA were as or more effective than DESS. Conversely, in cases where DESS performed more poorly, none of the six DESS-variant storage solutions provided better protection of HMW DNA than DESS. Moreover, for all taxa and storage intervals longer than one day, tissues stored in solutions containing DMSO alone, NaCl alone or DMSO and NaCl in combination resulted in %R and nY of HMW DNA significantly lower than those of fresh tissues. These results indicate that for the taxa, solutions and time intervals examined, only EDTA contributed directly to preservation of high molecular weight DNA.


Assuntos
DNA/química , Ácido Edético/química , Ácido Edético/farmacologia , Preservação de Tecido/métodos , Animais , Composição de Medicamentos , Peso Molecular
18.
Mol Cell ; 80(1): 102-113.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853547

RESUMO

Repair of covalent DNA-protein crosslinks (DPCs) by DNA-dependent proteases has emerged as an essential genome maintenance mechanism required for cellular viability and tumor suppression. However, how proteolysis is restricted to the crosslinked protein while leaving surrounding chromatin proteins unharmed has remained unknown. Using defined DPC model substrates, we show that the DPC protease SPRTN displays strict DNA structure-specific activity. Strikingly, SPRTN cleaves DPCs at or in direct proximity to disruptions within double-stranded DNA. In contrast, proteins crosslinked to intact double- or single-stranded DNA are not cleaved by SPRTN. NMR spectroscopy data suggest that specificity is not merely affinity-driven but achieved through a flexible bipartite strategy based on two DNA binding interfaces recognizing distinct structural features. This couples DNA context to activation of the enzyme, tightly confining SPRTN's action to biologically relevant scenarios.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , Linhagem Celular , Proteínas de Ligação a DNA/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Domínios Proteicos , Relação Estrutura-Atividade
19.
Nat Protoc ; 15(9): 3064-3087, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807907

RESUMO

Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a 'green' alternative known as RNAi, which involves the delivery of siRNAs that downregulate endogenous genes to confer resistance. However, siRNA is a molecule that is highly susceptible to enzymatic degradation and is difficult to deliver across the lignin-rich and multi-layered plant cell wall that poses the dominant physical barrier to biomolecule delivery in plants. We have demonstrated that DNA nanostructures can be utilized as a cargo carrier for direct siRNA delivery and gene silencing in mature plants. The size, shape, compactness and stiffness of the DNA nanostructure affect both internalization into plant cells and subsequent gene silencing efficiency. Herein, we provide a detailed protocol that can be readily adopted with standard biology benchtop equipment to generate geometrically optimized DNA nanostructures for transgene-free and force-independent siRNA delivery and gene silencing in mature plants. We further discuss how such DNA nanostructures can be rationally designed to efficiently enter plant cells and deliver cargoes to mature plants, and provide guidance for DNA nanostructure characterization, storage and use. The protocol described herein can be completed in 4 d.


Assuntos
DNA/química , Portadores de Fármacos/química , Engenharia , Nanoestruturas/química , RNA Interferente Pequeno/metabolismo , Tabaco/metabolismo , DNA/metabolismo , Portadores de Fármacos/metabolismo , RNA Interferente Pequeno/genética , Tabaco/genética
20.
Nat Protoc ; 15(9): 3030-3063, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32807909

RESUMO

Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid-responsive materials using the CRISPR-associated nuclease Cas12a as a user-programmable sensor and material actuator. This approach improves on the sensitivity of current DNA-responsive materials while enabling their rapid repurposing toward new sequence targets. Here, we provide a comprehensive resource for the design, synthesis and actuation of CRISPR-responsive hydrogels. First, we provide guidelines for the synthesis of Cas12a guide RNAs (gRNAs) for in vitro applications. We then outline methods for the synthesis of both polyethylene glycol-DNA (PEG-DNA) and polyacrylamide-DNA (PA-DNA) hydrogels, as well as their controlled degradation using Cas12a for the release of cargos, including small molecules, enzymes, nanoparticles and living cells within hours. Finally, we detail the design and assembly of microfluidic paper-based devices that use Cas12a-sensitive hydrogels to convert DNA inputs into a variety of visual and electronic readouts for use in diagnostics. Following the initial validation of the gRNA and Cas12a components (1 d), the synthesis and testing of either PEG-DNA or PA-DNA hydrogels require 3-4 d of laboratory time. Optional extensions, including the release of primary human cells or the design of the paper-based diagnostic, require an additional 2-3 d each.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas e Procedimentos Diagnósticos , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Materiais Inteligentes/química , Resinas Acrílicas/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/química , DNA/genética , Endodesoxirribonucleases/metabolismo , Humanos , Células K562 , Polietilenoglicóis/química , RNA Guia/genética
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