Name: Folding and Probing of Nucleic Acid Constructs
Uploaded: 2023-05-12T14:40:00
Description: Watch this Scientific Journal Video about Folding and Probing of Nucleic Acid Constructs at JoVE.com

folding and probing of nucleic acid constructs

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.

Watch this Scientific Journal Video about Folding and Probing of Nucleic Acid Constructs at JoVE.com

Folding and Probing of Nucleic Acid Constructs  

핵산 구조체의 접힘 및 프로빙

G4-TBA, 단일 가닥 TBA 및 이중 가닥 TBA 용액을 접을 수 있도록 3개의 서로 다른 튜브에 피펫팅하는 것으로 시작합니다. 샘플을 섭씨 95도까지 5분 동안 가열합니다. 실온으로 천천히 식히십시오.튜브가 실온으로 냉각되면 튜브를 원심분리합니다. 다음으로, 7개의 빈 오토클레이브 0.5밀리리터 튜브를 각각 3세트씩 구성합니다. 각 튜브에 3마이크로리터의 초순수를 추가합니다.접힌 핵산 구조체 5 마이크로리터를 세트의 각 튜브에 피펫팅합니다. 다음으로, 25 또는 250 마이크로 몰 B-CEP1의 2 마이크로 리터를 첨가하여 각각 5 및 50 마이크로 몰의 최종 프로브 농도를 얻습니다. 3개의 대조 튜브에 화합물 대신 초순수를 추가하고 모든 샘플을 섭씨 37도에서 배양합니다.튜브를 섭씨 20도에 두어 반응을 중지한 다음 진공 원심분리기에서 샘플을 건조시킵니다.

folding-and-probing-of-nucleic-acid-constructs

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JoVE Journal

Biochemistry

Folding and Probing of Nucleic Acid Constructs

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

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.

Separation of Polymerized Nucleic Acid Constructs by PAGE 

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.