separation of polymerized nucleic acid constructs by page 

Chemical Mapping을 사용하여 DNA 템플릿에서 G-Quadruplexes 식별

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.

저자 스포트라이트: Bis-3-Chloropiperidine 기반 화학 매핑에 의한 DNA G-Quadruplex 특성화 

In Vitro (시험관) Bis-3-Chloropiperidines에 의한 G-Quadruplex DNA 구조의 화학적 매핑

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   

PAGE에 의한 중합 핵산 조성물의 분리 

중합된 페이지 젤 샌드위치를 준비하는 것으로 시작합니다. 클램프와 종이 테이프 층을 제거하고 빗을 천천히 제거합니다. 증류수로 우물을 철저히 헹굽니다.젤 샌드위치를 수직 전기 영동 장치에 올바르게 놓습니다. 상단 및 하단 저장소를 TBE 버퍼로 채웁니다. 50와트에서 최소 30분 동안 겔 전기영동을 사전 실행하여 플레이트를 예열합니다.한편, 건조된 샘플을 5마이크로리터의 변성 겔 로딩 완충액에 다시 현탁시킵니다. 다음으로, TBE 완충액으로 채워진 작은 주사기를 사용하여 겔의 웰에서 요소를 씻어냅니다. 이제 샘플을 깨끗한 우물에 넣고 적재 냄새를 기록해 둡니다.50와트에서 2시간 동안 또는 브로모페놀 블루 염료가 겔의 2/3를 소모할 때까지 겔을 실행합니다. 전기영동 후 전원을 끄고 유리 샌드위치를 조심스럽게 제거한 다음 유리판을 청소하십시오. 겔을 겔 이미저에 넣어 FAM 표지 올리고뉴클레오티드 띠의 형광을 검출합니다.1시간, 4시간 및 15시간 배양 후, 5 또는 50 마이크로몰 Bsep 1을 G4 TBA, 단일 가닥 TBA 및 이중 가닥 TBA의 2개의 마이크로몰 샘플과 반응시켰다. 각 샘플 세트에 대해 컨트롤이 로드되었습니다. G4 TBA 기질은 알킬화 및 후속 좌초 절단을 위한 G8의 가용성으로 인해 하나의 알킬화 부가물만 나타냈습니다.단일 및 이중 가닥 TBA에서 분할 산물의 다중 부가물 및 밴드가 관찰되었는데, 이는 모든 구아닌이 Bsep 반응에 취약했기 때문입니다. 프로빙 반응 및 트리스 완충액은 원치 않는 친핵체의 존재로 인해 비효율적인 화학적 매핑을 초래하여 고유 활성을 감소시켰습니다. G4 TBA 샘플은 좌초 절단 없이 부가물의 형성만을 보여주었습니다.단일 및 이중 가닥 TBA의 경우 24시간 배양만으로도 DNA 단편화가 발생했으며, 이는 Tris가 없는 15시간 단편화와 비슷합니다.

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Biochemistry

Separation of Polymerized Nucleic Acid Constructs by PAGE 

161 조회수.  University of Padova. 중합된 페이지 젤 샌드위치를 준비하는 것으로 시작합니다. 클램프와 종이 테이프 층을 제거하고 빗을 천천히 제거합니다. 증류수로 우물을 철저히 헹굽니다.젤 샌드위치를 수직 전기 영동 장치에 올바르게 놓습니다. 상단 및 하단 저장소를 TBE 버퍼로 채웁니다. 50와트에서 최소 30분 동안 겔 전기영동을 사전 실행하여 플레이트를 예열합니다.한편, 건조된 샘플을 5...

비디오: 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 ...

연구

생화학

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.

핵산 구조체의 접힘 및 프로빙

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.