separation of polymerized nucleic acid constructs by page 

使用化学作图鉴定 DNA 模板中的 G-四链体

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.

作者聚焦：通过基于 Bis-3-氯哌啶的化学定位表征 DNA G-四链体 

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

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.

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Separation of Polymerized Nucleic Acid Constructs by PAGE   

通过PAGE分离聚合核酸构建体 

首先准备聚合页面凝胶三明治。取下夹子和纸胶带层，慢慢取下梳子。用蒸馏水彻底冲洗孔。将凝胶夹层正确放置在垂直电泳装置中。用TBE缓冲液填充顶部和底部储液罐。通过在 50 瓦的功率下进行凝胶电泳预运行至少 30 分钟来预热板。同时，将干燥的样品重悬于5微升变性凝胶上样缓冲液中。接下来，使用装有TBE缓冲液的小注射器将尿素从凝胶孔中冲洗出来。现在将样品加载到干净的孔中，注意加载的气味。以 50 瓦的功率运行凝胶两个小时，或直到溴酚蓝染料耗尽凝胶的 2/3。电泳后，关闭电源，小心取出玻璃夹层，清洗玻璃板。将凝胶置于凝胶成像仪中以检测FAM标记的寡核苷酸条带的荧光。孵育 1、4 和 15 小时后，5 或 50 微摩尔 Bsep 1 与 G4 TBA、单链 TBA 和双链 TBA 的 2 微摩尔样品反应。为每组样品加载对照。G4 TBA 底物仅显示一个烷基化加合物，因为只有 G8 可用于烷基化和随后的链状裂解。在单链和双链TBA中观察到多个加合物和裂解产物条带，因为所有鸟嘌呤都对Bsep反应敏感。由于存在不需要的亲核试剂，探测反应和tris缓冲液导致化学图谱分析效率低下，导致本体活性降低。G4 TBA样品仅显示加合物的形成，而没有链状裂解。对于单链和双链 TBA，仅孵育 24 小时就会导致 DNA 片段化，与没有 Tris 的 15 小时片段化相当。

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

Research

JoVE Journal

Biochemistry

Separation of Polymerized Nucleic Acid Constructs by PAGE 

161 次观看.  University of Padova. 首先准备聚合页面凝胶三明治。取下夹子和纸胶带层，慢慢取下梳子。用蒸馏水彻底冲洗孔。将凝胶夹层正确放置在垂直电泳装置中。用TBE缓冲液填充顶部和底部储液罐。通过在 50 瓦的功率下进行凝胶电泳预运行至少 30 分钟来预热板。同时，将干燥的样品重悬于5微升变性凝胶上样缓冲液中。接下来，使用装有TBE缓冲液的小注射器将尿素从凝胶孔中冲洗出来。现在...

视频: 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 ...

科研

生物化学

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.

核酸构建体的折叠和探测

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.