Name: Separation of Polymerized Nucleic Acid Constructs by PAGE
Uploaded: 2023-05-12T14:40:00
Description: 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 

تحديد رباعيات G في قالب الحمض النووي باستخدام رسم الخرائط الكيميائية

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.

تسليط الضوء على المؤلف: توصيف الحمض النووي G-quadruplex بواسطة رسم الخرائط الكيميائية القائمة على Bis-3-Chloropiperidine 

في المختبر رسم الخرائط الكيميائية لهياكل الحمض النووي G-quadruplex بواسطة Bis-3-chloropiperidines

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   

فصل تركيبات الحمض النووي المبلمرة بواسطة PAGE 

ابدأ بإعداد شطيرة جل مبلمرة. قم بإزالة المشابك وطبقات الشريط الورقي ، وقم بإزالة المشط ببطء. شطف جيدا الآبار بالماء المقطر.ضع شطيرة الجل بشكل صحيح في جهاز الكهربائي العمودي. املأ الخزانات العلوية والسفلية بمخزن TBE المؤقت. قم بتسخين الألواح عن طريق إجراء تشغيل مسبق للرحلان الكهربائي الهلامي لمدة 30 دقيقة على الأقل عند 50 واط.وفي الوقت نفسه ، أعد تعليق العينات المجففة في خمسة ميكرولترات من المخزن المؤقت لتحميل الجل الذي تم تغيير طبيعته. بعد ذلك ، استخدم حقنة صغيرة مملوءة بمحلول TBE لطرد اليوريا من آبار الجل. الآن قم بتحميل العينات في الآبار النظيفة ، مع ملاحظة رائحة التحميل.قم بتشغيل الجل لمدة ساعتين عند 50 واط أو حتى تنفد صبغة البروموفينول الزرقاء 2/3 من الجل. بعد الرحلان الكهربائي ، قم بإيقاف تشغيل مصدر الطاقة ، وقم بإزالة الساندويتش الزجاجي بعناية ، وتنظيف الألواح الزجاجية. ضع الجل في جهاز تصوير هلامي للكشف عن مضان نطاقات قليل النوكليوتيد التي تحمل علامة FAM.بعد ساعة واحدة وأربع و 15 ساعة من الحضانة ، تفاعلت إما خمس أو 50 ميكرومولار Bsep 1 مع عينتين ميكرومولار من G4 TBA ، TBA أحادي الشريط ، و TBA مزدوج تقطعت بهم السبل. تم تحميل عناصر التحكم لكل مجموعة من العينات. أظهرت الركيزة G4 TBA تقريب ألكلة واحد فقط بسبب توفر G8 فقط للألكلة والانقسام اللاحق الذين تقطعت بهم السبل.لوحظت العديد من المضافات والنطاقات من منتجات الانقسام في TBA مفرد ومزدوج تقطعت بهم السبل ، لأن جميع الجوانين كانت عرضة لتفاعل Bsep. أدى تفاعل التحقيق ومخزن تريس المؤقت إلى رسم خرائط كيميائية غير فعالة بسبب وجود النيوكليوفيليات غير المرغوب فيها ، مما تسبب في انخفاض نشاط الحس العميق. أظهرت عينات G4 TBA فقط تكوين التقريب دون انقسام تقطعت بهم السبل.بالنسبة ل TBA الفردي والمزدوج الذي تقطعت به السبل ، أدت الحضانة لمدة 24 ساعة فقط إلى تجزئة الحمض النووي ، مقارنة بالتجزئة لمدة 15 ساعة بدون Tris.

Research

JoVE Journal

Biochemistry

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 ...