separation of polymerized nucleic acid constructs by page 

Identificando G-Quadruplexes em um modelo de DNA usando mapeamento químico

In addition to the typical Watson-Crick double helix, nucleic acids can adopt various secondary structures, such as the alternative G-quadruplex (G4) form, due to their guanine-rich sequences. G4 structure is based on the formation of planar tetramers, called G-tetrads, in which four guanines interact through Hoogsteen hydrogen bonds. G-tetrads are stacked and further stabilized by monovalent cations that are coordinated in the center of the guanine core (Figure 1)1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65373/65373fig01.jpg" /> 
Figure 1: Schematic representation of a G-quadruplex structure. (A) Schematic representation of a G-tetrad. The planar array is stabilized by Hoogsteen base-pairing and by a central cation (M+).&#160;<a href="https://www.jove.com/files/ftp_upload/65373/65373fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sequences with four or more runs of at least two consecutive guanine nucleotides are potential G-quadruplex-forming sequences (PQSs) that can fold in G-quadruplex structures. PQSs are located in many different cellular contexts, such as at telomeres, gene promoters, ribosomal DNA, and recombination sites, and are involved in the regulation of many biological processes2. Hence, the identification and experimental validation of G4s in the human genome, which is currently performed primarily through computational tools, is a biologically relevant issue3. In order to support computational predictions or detect unpredicted G4 structures, an accessible method based on chemical mapping to identify the G4 formation in a DNA template is shown here, enabling the precise identification of guanines forming the G-tetrad structure.
The reported chemical mapping assay exploits the different reactivity of bis-3-chloropiperidines (B-CePs) with guanines following the formation of G4 structures. Due to their high reactivity with nucleophiles4,5,6,7,8,9, B-CePs are nucleic acid-alkylating agents with the ability to react very efficiently with the N7 position of guanine nucleotides10. Alkylation is followed by depurination and strand cleavage in single- and double-stranded DNA constructs. On the contrary, guanines involved in the formation of the G-tetrads in G4 arrangements are impervious to B-CeP alkylation, as the N7 position of guanines is implicated in the Hoogsteen hydrogen bonds. This specific reactivity of B-CePs allows not only the detection of G4 structures, but also the identification of the guanines forming the tetrad(s), as they can be deduced from their relative protection from alkylation compared with guanines in single- and double-stranded DNA.
The chemical mapping protocol is reported here using B-CeP 1 (Figure 2A) as a probe for the characterization of thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement in the presence of potassium cations11,12. The G4 arrangement of TBA (G4-TBA) is directly compared with two controls, namely TBA in the single-stranded form (ssTBA) and TBA annealed to its complementary sequence to form the double-stranded construct (dsTBA) (Table 1). Products of probing reactions are resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at the single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Visualization on the gel is enabled by conjugation of the TBA oligonucleotide with a fluorophore at its 3&#39;-end (Table 1). This protocol shows how to fold TBA in its different conformations (G4 and controls), and how to perform probing reactions with B-CePs followed by PAGE.

Autor em destaque: Caracterizando o DNA G-Quadruplex por mapeamento químico baseado em bis-3-cloropiperidina 

In vitro Mapeamento químico de estruturas do DNA G-Quadruplex por bis-3-cloropiperidinas;

G-quadruplex are nucleic acid secondary structures that typically formed within guanine-rich DNA sequences. The chemical mapping of G4 by B-CePs aims at the in vitro characterization of G4, and allows the identification of the specific guanines forming the G-tetrads. The identification and the experimental validation of G4s within potential G-quadruplex-forming sequences is a biologically relevant issue, which is currently addressed primary through computational tools.To support such predictions and detect the unpredicted G4, chemical mapping by B-CePs represents an accessible method to identify G4 in DNA sequences. The chemical mapping by B-CePs is a convenient alternative to the widely used dimethyl sulfate footprinting protocol for the characterization of G-quadruplex. The key advantage by B-CePs is that it reduces the number of steps in the protocol and it reduces the initial amount of the DNA substrate.The chemical mapping by B-CePs is described here with the TBA sequence as a proof of concept. The protocol will lead to new experiments applicable to any other potential G-quadruplex-forming sequence exploring both DNA and RNA constructs.

Watch this Scientific Journal Video about Separation of Polymerized Nucleic Acid Constructs by PAGE at JoVE.com

Separation of Polymerized Nucleic Acid Constructs by PAGE   

Separação de Construções de Ácidos Nucleicos Polimerizados por PAGE 

Comece preparando um sanduíche de gel de página polimerizado. Retire as abraçadeiras e as camadas de fita de papel e remova lentamente o pente. Enxágue bem os poços com água destilada.Coloque o sanduíche de gel corretamente no aparelho de eletroforese vertical. Encha os reservatórios superior e inferior com tampão TBE. Aqueça as placas realizando uma pré-execução da eletroforese em gel por pelo menos 30 minutos a 50 watts.Enquanto isso, ressuspenda as amostras secas em cinco microlitros de tampão de carregamento de gel desnaturante. Em seguida, use uma pequena seringa cheia de tampão TBE para liberar a ureia para fora dos orifícios do gel. Agora carregue as amostras nos poços limpos, anotando o odor do carregamento.Passe o gel por duas horas a 50 watts ou até que o corante azul de bromofenol tenha escorrido 2/3 do gel. Após a eletroforese, desligue a fonte de alimentação, remova cuidadosamente o sanduíche de vidro e limpe as placas de vidro. Coloque o gel em um imageador de gel para detectar a fluorescência das bandas de oligonucleotídeos marcadas com FAM.Após uma, quatro e 15 horas de incubação, cinco ou 50 Micromolares Bsep 1 reagiram com duas amostras micromolares de TBA G4, TBA de fita simples e TBA de fita dupla. Os controles foram carregados para cada conjunto de amostras. O substrato G4 TBA apresentou apenas um aduto de alquilação devido à disponibilidade de apenas G8 para alquilação e posterior clivagem encalhada.Múltiplos adutos e bandas de produtos de clivagem foram observados em TBA de fita simples e dupla, pois todas as guaninas foram suscetíveis à reação de Bsep. A reação de sondagem e o tampão tris resultaram em mapeamento químico ineficiente devido à presença de nucleófilos indesejados, causando redução da proprioatividade. As amostras de TBA G4 mostraram apenas a formação de aduto sem clivagem encalhada.Para TBA de fita simples e dupla, apenas uma incubação de 24 horas levou à fragmentação do DNA, comparável à fragmentação de 15 horas sem Tris.

separation-of-polymerized-nucleic-acid-constructs-by-page

Research

JoVE Journal

Biochemistry

Separation of Polymerized Nucleic Acid Constructs by PAGE 

161 visualizações.  University of Padova. Comece preparando um sanduíche de gel de página polimerizado. Retire as abraçadeiras e as camadas de fita de papel e remova lentamente o pente. Enxágue bem os poços com água destilada.Coloque o sanduíche de gel corretamente no aparelho de eletroforese vertical. Encha os reservatórios superior e inferior com tampão TBE. Aqueça as placas realizando uma pré-execução da eletroforese em gel por pelo menos 30 minutos a 50 watts.Enquanto isso, ressuspenda as amostras secas...

Vídeo: Separation of Polymerized Nucleic Acid Constructs by PAGE   

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

Bioquímica

Folding and Probing of Nucleic Acid Constructs

Begin by pipetting G4-TBA, single-stranded TBA, and double-stranded TBA solutions into three different tubes for folding. Heat the samples to 95 degrees Celsius for five minutes. Let them cool slowly to room temperature.Once the tubes have cooled to room temperature, centrifuge the tubes. Next, organize three sets each of seven empty autoclaved 0.5-milliliter tubes. Add three microliters of ultrapure water into each tube.Pipette five microliters of the folded nucleic acid constructs into each tube of the set. Next, add two microliters of either 25 or 250-micromolar B-CEP1 to obtain the final probe concentration of five and 50 micromolar respectively. To the three control tubes, add ultrapure water instead of the compound and incubate all the samples at 37 degrees Celsius.Stop the reaction by placing the tubes at 20 degrees Celsius, then dry the samples in a vacuum centrifuge.

Dobramento e sondagem de construções de ácidos nucleicos

Begin by preparing a polymerized page gel sandwich. Remove the clamps and paper tape layers, and slowly remove the comb. Thoroughly rinse the wells with distilled water.Place the gel sandwich correctly in the vertical electrophoresis apparatus. Fill the top and bottom reservoirs with TBE buffer. Warm up the plates by performing a pre-run of the gel electrophoresis for at least 30 minutes at 50 watts.Meanwhile, re-suspend the dried samples in five microliters of denaturing gel loading buffer. Next, use a small syringe filled with TBE buffer to flush the urea out of the wells of the gel. Now load the samples into the clean wells, taking note of the odor of loading.Run the gel for two hours at 50 watts or until the bromophenol blue dye has run down 2/3 of the gel. After electrophoresis, turn off the power supply, carefully remove the glass sandwich, and clean the glass plates. Place the gel in a gel imager to detect the fluorescence of the FAM-labeled oligonucleotide bands.After one, four, and 15 hours of incubation, either five or 50 Micromolar Bsep 1 reacted with two micromolar samples of G4 TBA, single-stranded TBA, and double-stranded TBA. Controls were loaded for each set of samples. The G4 TBA substrate showed only one alkylation adduct due to the availability of only G8 for alkylation and subsequent stranded cleavage.Multiple adducts and bands of cleavage products were observed in single and double-stranded TBA, because all guanines were susceptible to Bsep reaction. Probing reaction and tris buffer resulted in inefficient chemical mapping due to the presence of undesired nucleophiles, causing reduced proprioactivity. The G4 TBA samples showed only the formation of adduct without stranded cleavage.For the single and double-stranded TBA, only a 24-hour incubation led to DNA fragmentation, comparable to the 15-hour fragmentation without Tris.