This work was supported by the Department of Pharmaceutical and Pharmacological Sciences, University of Padova (PRIDJ-BIRD2019).

1. Nucleic acid and chemical probe preparation
<ol>
	<li>Nucleic acids 
	NOTE: The oligonucleotide named &#34;TBA&#34; is the 15-mer DNA sequence 5&#39;-GGT-TGG-TGT-GGT-TGG-3&#39; labeled at the 3&#39;-end by the fluorophore 5-carboxyfluorescein (FAM) to enable visualization on the gel. The unlabeled oligonucleotide &#34;cTBA&#34; is its DNA complementary sequence 5&#39;-CCA-ACC-ACA-CCA-ACC-3&#39;. TBA and cTBA are employed to obtain the three different structures, as shown in Table 1.
	<ol>
		<li>Autoclave tips and 0.5 mL tubes in order to obtain sterile disposables and avoid contamination.</li>
		<li>Prepare stock solutions solubilizing each oligonucleotide in ultrapure water to a final concentration of 100 &#181;M. Determine the exact oligonucleotide concentration with a ultraviolet-visible (UV-Vis) spectrophotometer, using the extinction coefficient at 260 nm provided by the manufacturer. 
		NOTE: Extinction coefficients: 164,300 M-1 cm-1 and 138,600 M-1 cm-1 were used for TBA and cTBA, respectively.</li>
		<li>Store TBA and cTBA stock solutions at -20 &#176;C (for months in these conditions).</li>
	</ol>
	</li>
	<li>Compound B-CeP 1 
	NOTE: The compound B-CeP 1 is synthesized as previously reported6.
	<ol>
		<li>Prepare the B-CeP 1 stock solution at ~10 mM. Weigh ~1 mg of the lyophilized compound using an analytical balance located in a fume hood and solubilize it in 100% dimethyl sulfoxide (DMSO).</li>
		<li>Calculate the exact compound concentration based on the actual amount of compound and DMSO (d = 1.1 g/cm3) used. 
		NOTE: Handle the compound with gloves at all times (both when lyophilized and when dissolved in DMSO)13,14.</li>
	</ol>
	</li>
</ol>
Table 1: Oligonucleotide structures used in this protocol. <a href="https://www.jove.com/files/ftp_upload/65373/65373_Table 1 (5).xlsx" target="_blank">Please click here to download this Table.</a>
2. Folding of nucleic acid constructs
<ol>
	<li>Preparation of buffers
	<ol>
		<li>Prepare a BPE buffer solution (biphosphate-ethylenediaminetetraacetic acid [EDTA], 5x: 2 mM NaH2PO4, 6 mM Na2HPO4, 1 mM Na2EDTA, pH 7.4) and a solution of 500 mM KCl in ultrapure water. Filter the solutions through 0.22 &#181;m pore size filters. 
		NOTE: For best results, use freshly prepared solutions. BPE buffer can be stored at 4 &#176;C for up to 15 days.</li>
	</ol>
	</li>
	<li>Folding of G4-TBA, ssTBA, and dsTBA samples by the heat-refolding procedure 
	NOTE: Potassium cations are necessary to fold the G4 structure (G4-TBA). Do not add potassium cations in the folding solution of the controls ssTBA and dsTBA.
	<ol>
		<li>Prepare 40 &#181;L of a 4 &#181;M solution of G4-TBA in 1x BPE and 100 mM KCl. Denature the oligonucleotide G4-TBA solution by heating the tube to 95 &#176;C for 5 min and slowly cool it down to room temperature (RT) to allow the TBA to fold into G-quadruplexes.</li>
		<li>Prepare 40 &#181;L of a 4 &#181;M solution of ssTBA in 1x BPE. Perform the heat-refolding procedure, as mentioned in step 2.2.1, to fold TBA in its single-stranded form.</li>
		<li>Prepare 40 &#181;L of a 4 &#181;M solution of dsTBA by mixing equimolar amounts of TBA and cTBA in 1x BPE. Perform the heat-refolding procedure as mentioned above (step 2.2.1) for TBA to anneal to its complementary sequence cTBA and form the double-stranded form of TBA. 
		NOTE: The final volume of each folding solution is based on the number of samples for the probing reactions, considering that 5 &#181;L of 4 &#181;M solution will be needed for each sample. Prepare a little excess volume of each solution to avoid pipetting errors.</li>
	</ol>
	</li>
</ol>
3. Probing reactions
NOTE: Probing reactions must be done immediately after the heat-refolding procedure.
<ol>
	<li>When the folding solutions of G4-TBA, ssTBA, and dsTBA have cooled to RT, perform a short-spin centrifugation (7,000 &#215; g for 5-8 s at RT) and start the probing reactions.</li>
	<li>Prepare 21 empty 0.5 mL autoclaved tubes. Organize them in three sets of seven tubes each in the rack for lab samples, as reported in Table 2. 
	NOTE: Each column set corresponds to the three different TBA folding conditions G4-TBA, ssTBA, and dsTBA. Each row corresponds to three different incubation times. Each cell within the column corresponds to the final B-CeP 1 probe concentration (Table 2). Make sure to clearly label the tubes.</li>
	<li>Add 3 &#181;L of ultrapure water in each tube.</li>
	<li>Add 5 &#181;L of folded G4-TBA in each tube of the first set (step 3.2). Add 5 &#181;L of folded ssTBA in each tube of the second set. Add 5 &#181;L of folded dsTBA in each tube of the third set.</li>
	<li>Dilute the B-CeP 1 stock solution to 250 &#181;M and 25 &#181;M in ultrapure water. 
	NOTE: Dilutions of the B-CeP 1 chemical probe must be freshly prepared and immediately reacted with the DNA substrate to avoid competing reactions with water.</li>
	<li>Add 2 &#181;L of the appropriate B-CeP 1 dilution (25 &#181;M and 250 &#181;M) to the samples. Replace the compound with ultrapure water in the three control samples (C) for analysis of the differently folded TBAs in the absence of the compound. Incubate all the samples at 37 &#176;C.</li>
	<li>After 1 h, 4 h, and 15 h of incubation, stop the reaction by placing the tubes at -20 &#176;C until the next step. 
	NOTE: The samples can be stored in these conditions for a couple of days.</li>
	<li>Dry the samples in a vacuum centrifuge. 
	NOTE: The dried samples can be stored at -20 &#176;C for weeks before proceeding with the PAGE analysis.</li>
</ol>
Table 2: Samples for the probing reactions (structures, probe concentrations, and incubation time). Each column set corresponds to the three different TBA folding conditions (G4-TBA, ssTBA, and dsTBA). Each row corresponds to three different incubation times (1, 4, 15 h). Each cell within the column corresponds to the final B-CeP 1 probe concentration (5 or 50 &#181;M). The control (C) for each set corresponds to a sample of the differently folded TBAs incubated for the longer time (15 h) in the absence of compound. <a href="https://www.jove.com/files/ftp_upload/65373/65373_Table 2 (3).xlsx" target="_blank">Please click here to download this Table.</a>
4. High-resolution PAGE
<ol>
	<li>Preparation of the denaturing polyacrylamide solution 
	NOTE: Prepare in advance 500 mL of 20% denaturing polyacrylamide gel solution. Around 80 mL of this solution will be used for each experiment. Use an amber glass bottle or cover a glass bottle with aluminum foil to store the solution at RT. 
	CAUTION: Polyacrylamide is neurotoxic. During all steps of gel preparation and pouring, wear gloves and a lab coat. Discard polymerized acrylamide in an appropriate box for contaminated materials.
	<ol>
		<li>Weigh 210 g of urea in a 1 L beaker. Add 250 mL of 40% acrylamide/bisacrylamide (19/1) solution and 50 mL of 10x TBE (890 mM Tris-HCl, 890 mM borate, 20 mM EDTA, pH 8).</li>
		<li>Place the beaker on a stir plate and mix the solution with a stirring rod. Cover the beaker with aluminum foil during mixing to prevent splashing and contamination.</li>
		<li>Mix the solution until the urea is completely dissolved and the solution is clear. 
		NOTE: This step can take many hours. To promote the dissolution of urea, add a small aliquot of water without going beyond the desired final volume.</li>
		<li>Remove the stirring rod. Pour the solution in a cylinder and add water to an exact final volume of 500 mL.</li>
	</ol>
	</li>
	<li>Setting up of gel apparatus
	<ol>
		<li>Clean two plates (one notched and one unnotched) with 70% ethanol, let them dry, and then treat the plates with a dimethyldichlorosilane solution. 
		NOTE: Silanization can be skipped, although it helps the release of the gel from one of the plates when the gel sandwich is taken apart. 
		CAUTION: Handle the silanization solution with gloves and carry out the plate treatment with this solution in a fume hood.</li>
		<li>Place the 0.4 mm spacers along the long edges of the longer plate, place the short plate on top of the other, and align the two plates at the bottom.</li>
		<li>Place multiple layers of paper tape along all the edges except for the top.</li>
		<li>To avoid leaks during casting, add an additional layer of tape to the bottom of the gel.</li>
		<li>Clip the sides of the glass sandwich with clean clamps following the supplier&#39;s instructions (different suppliers use slightly different apparatus, sandwich clamps, and gaskets).</li>
	</ol>
	</li>
	<li>Pouring the gel 
	NOTE: Pour the gel at RT (25 &#176;C), since polyacrylamide polymerization is temperature-sensitive.
	<ol>
		<li>In a beaker, pour 80 mL of the previously prepared denaturing polyacrylamide solution (step 4.1), 450 &#181;L of a 10% m/V ammonium persulphate (APS) solution, and 45 &#181;L of tetramethylethylenediamine (TEMED) immediately before use.</li>
		<li>Mix the solution and rapidly pour it down between the glass plates using a 50 mL syringe. Introduce the comb with the desired number of wells between the glass plates, avoiding bubbles. Add gel solution to fill the sandwich completely, if necessary. Place four clamps on the comb to press down and allow for even distribution of the wells.</li>
		<li>Let the gel polymerize for at least 45 min.</li>
	</ol>
	</li>
	<li>Running the gel
	<ol>
		<li>After the polymerization, remove all the clamps and paper tape layers. Remove the comb slowly and thoroughly rinse the wells with distilled water.</li>
		<li>Follow the instructions from the specific supplier to correctly place the gel sandwich in the vertical gel electrophoresis apparatus.</li>
		<li>Prepare TBE running buffer (1x: 89 mM Tris-HCl, 89 mM borate, 2 mM EDTA, pH 8) in deionized water and fill both the top and bottom reservoirs with the buffer.</li>
		<li>Warm up the plates by performing a pre-run of the gel electrophoresis for at least 30 min at 50 W.</li>
		<li>Prepare denaturing gel loading buffer (DGLB: 1 M Tris-HCl, 80% formamide, 50% glycerol, 0.05% bromophenol blue) in ultrapure water. 
		NOTE: GLB helps to track the oligonucleotide samples&#39; movement into the gel system and allows to load the samples into the gel&#39;s wells. The presence of the denaturing agent formamide allows DNA species to separate according to size, even in a non-denaturing PAGE.</li>
		<li>Resuspend the dried samples (samples from step 3.8) in 5 &#181;L of DGLB.</li>
		<li>Before loading the samples, clean the wells using a small syringe and the TBE buffer in the upper buffer chamber to remove the urea from the wells. 
		NOTE: Repeat this step several times to accurately clean the wells, thus avoiding bands difficult to be interpreted.</li>
		<li>Load the samples into the clean wells and make a note of the order of loading.</li>
		<li>Run the gel electrophoresis for 2 h at 50 W, or at least until the bromophenol blue dye has run 2/3 down the gel.</li>
	</ol>
	</li>
	<li>Imaging the gel
	<ol>
		<li>After electrophoresis, turn off the power supply, remove the glass sandwich, and clean the glasses.</li>
		<li>Detect the fluorescence of the FAM-labeled oligonucleotide bands by scanning using a gel imager.</li>
	</ol>
	</li>
</ol>

Identifying G-Quadruplexes in a DNA Template Using Chemical Mapping

<ol><li>Davis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G-quartets+40+years+later%3A+from+5%27-GMP+to+molecular+biology+and+supramolecular+chemistry%5BTitle%5D)+AND+%22Angewandte+Chemie%22%5BJournal%5D)">G-quartets 40 years later: from 5'-GMP to molecular biology and supramolecular chemistry.</a> Angewandte Chemie. 43 (6), 668-698 (2004).</li>
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<li>Chambers, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+sequencing+of+DNA+G-quadruplex+structures+in+the+human+genome%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">High-throughput sequencing of DNA G-quadruplex structures in the human genome.</a> Nature Biotechnology. 33 (8), 877-881 (2015).</li>
<li>Zuravka, I., Sosic, A., Gatto, B., Gottlich, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+and+evaluation+of+a+bis-3-chloropiperidine+derivative+incorporating+an+anthraquinone+pharmacophore%5BTitle%5D)+AND+%22Bioorganic+%26+Medicinal+Chemistry+Letters%22%5BJournal%5D)">Synthesis and evaluation of a bis-3-chloropiperidine derivative incorporating an anthraquinone pharmacophore.</a> Bioorganic & Medicinal Chemistry Letters. 25 (20), 4606-4609 (2015).</li>
<li>Zuravka, I., Roesmann, R., Sosic, A., Gottlich, R., Gatto, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bis-3-chloropiperidines+containing+bridging+lysine+linkers%3A+Influence+of+side+chain+structure+on+DNA+alkylating+activity%5BTitle%5D)+AND+%22Bioorganic+%26+Medicinal+Chemistry%22%5BJournal%5D)">Bis-3-chloropiperidines containing bridging lysine linkers: Influence of side chain structure on DNA alkylating activity.</a> Bioorganic & Medicinal Chemistry. 23 (6), 1241-1250 (2015).</li>
<li>Zuravka, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+and+DNA+cleavage+activity+of+bis-3-chloropiperidines+as+alkylating+agents%5BTitle%5D)+AND+%22ChemMedChem%22%5BJournal%5D)">Synthesis and DNA cleavage activity of bis-3-chloropiperidines as alkylating agents.</a> ChemMedChem. 9 (9), 2178-2185 (2014).</li>
<li>Sosic, A., Gottlich, R., Fabris, D., Gatto, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((B-CePs+as+cross-linking+probes+for+the+investigation+of+RNA+higher-order+structure%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">B-CePs as cross-linking probes for the investigation of RNA higher-order structure.</a> Nucleic Acids Research. 49 (12), 6660-6672 (2021).</li>
<li>Sosic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bis-3-chloropiperidines+targeting+TAR+RNA+as+a+novel+strategy+to+impair+the+HIV-1+nucleocapsid+protein%5BTitle%5D)+AND+%22Molecules%22%5BJournal%5D)">Bis-3-chloropiperidines targeting TAR RNA as a novel strategy to impair the HIV-1 nucleocapsid protein.</a> Molecules. 26 (7), 1874(2021).</li>
<li>Sosic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+evaluation+of+bis-3-chloropiperidines+as+RNA+modulators+targeting+TAR+and+TAR-protein+interaction%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">In vitro evaluation of bis-3-chloropiperidines as RNA modulators targeting TAR and TAR-protein interaction.</a> International Journal of Molecular Sciences. 23 (2), 582(2022).</li>
<li>Sosic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+and+topoisomerase+II+mediated+DNA+damage+by+bis-3-chloropiperidines%3A+The+importance+of+being+an+earnest+G%5BTitle%5D)+AND+%22ChemMedChem%22%5BJournal%5D)">Direct and topoisomerase II mediated DNA damage by bis-3-chloropiperidines: The importance of being an earnest G.</a> ChemMedChem. 12 (17), 1471-1479 (2017).</li>
<li>Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H., Toole, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selection+of+single-stranded+DNA+molecules+that+bind+and+inhibit+human+thrombin%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Selection of single-stranded DNA molecules that bind and inhibit human thrombin.</a> Nature. 355 (6360), 564-566 (1992).</li>
<li>Paborsky, L. R., McCurdy, S. N., Griffin, L. C., Toole, J. J., Leung, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+single-stranded+DNA+aptamer-binding+site+of+human+thrombin%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">The single-stranded DNA aptamer-binding site of human thrombin.</a> The Journal of Biological Chemistry. 268 (28), 20808-20811 (1993).</li>
<li>Carraro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behind+the+mirror%3A+chirality+tunes+the+reactivity+and+cytotoxicity+of+chloropiperidines+as+potential+anticancer+agents%5BTitle%5D)+AND+%22ACS+Medicinal+Chemistry+Letters%22%5BJournal%5D)">Behind the mirror: chirality tunes the reactivity and cytotoxicity of chloropiperidines as potential anticancer agents.</a> ACS Medicinal Chemistry Letters. 10 (4), 552-557 (2019).</li>
<li>Carraro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appended+aromatic+moieties+in+flexible+bis-3-chloropiperidines+confer+tropism+against+pancreatic+cancer+cells%5BTitle%5D)+AND+%22ChemMedChem%22%5BJournal%5D)">Appended aromatic moieties in flexible bis-3-chloropiperidines confer tropism against pancreatic cancer cells.</a> ChemMedChem. 16 (5), 860-868 (2021).</li>
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The authors have no conflicts of interest to disclose.

G-quadruplexes are nucleic acid secondary structures that typically fold within guanine-rich DNA sequences, and are significant research targets because of their association with genetic control and diseases. Chemical mapping by B-CePs is a useful protocol for the characterization of DNA G4s, which can be used to identify the guanine bases involved in the formation of G-tetrads under physiological salt conditions.
The chemical probe used in this protocol is B-CeP 1 (Figure...

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.

<table><tbody><tr><td>Acrylamide/bis-acrylamide solution 40%</td><td>Applichem</td><td>A3658</td><td>R45-46-20/21-25-36/38-43-48/23/&lt;br/&gt; 24/25-62</td></tr><tr><td>Ammonium per-sulfate (APS)</td><td>Sigma Aldrich</td><td>A7460</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td></td><td></td></tr><tr><td>Autoclave</td><td>pbi international</td><td></td><td></td></tr><tr><td>Boric acid</td><td>Sigma Aldrich</td><td>B0252</td><td></td></tr><tr><td>Bromophenol blue Brilliant blue R</td><td>Sigma Aldrich</td><td>B0149</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate dodecahydrate</td><td>Fluka</td><td>71649</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>276855</td><td></td></tr><tr><td>DNA oligonucleotides</td><td>Integrated DNA Technologies</td><td>synthesis of custom sequences</td><td></td></tr><tr><td>EDTA disodium</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>Formamide</td><td>Fluka</td><td>40248</td><td>H351-360D-373</td></tr><tr><td>Gel imager</td><td>GE Healtcare</td><td>STORM B40</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td></td></tr><tr><td>Micro tubes 0.5 mL</td><td>Sarstedt</td><td>72.704</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Sequencing apparatus</td><td>Biometra</td><td>Model S2</td><td></td></tr><tr><td>Silanization solution I</td><td>Fluka</td><td>85126</td><td>H225, 314, 318, 336, 304, 400, 410</td></tr><tr><td>Sodium phosphate monobasic</td><td>Carlo Erba</td><td>480086</td><td></td></tr><tr><td>Speedvac concentrator</td><td>Thermo Scientific</td><td>Savant DNA 120</td><td></td></tr><tr><td>TEMED</td><td>Fluka</td><td>87689</td><td>R11-21/22-23-34</td></tr><tr><td>Tris-HCl</td><td>MERCK</td><td>1.08387.2500</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>51456</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Thermo Scientific</td><td>Nanodrop 1000</td><td></td></tr></tbody></table>

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.

Figure 2&#160;shows a representative result of a chemical mapping assay performed, as described in the protocol with B-CeP 1 on the TBA oligonucleotide folded in three different structures. The G-quadruplex arrangement of TBA (G4-TBA) was obtained by folding the oligonucleotide in BPE and in the presence of the K+ cation, whereas the single-stranded form of the same TBA sequence (ssTBA) was folded in the absence of potassium. The double-stranded construct (dsTBA) was prepared by a...

Watch this Scientific Journal Video about Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping at JoVE.com

Author Spotlight: Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping 

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.

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 ...

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Research

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Biochemistry

1.3K Views.  University of Padova. 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.

Video: Author Spotlight: Characterizing DNA G-Quadruplex by Bis-3-Chloropiperidine Based Chemical Mapping 

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Genetics

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Biology

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA &#39;roadblocks&#39;&#160;&#8212; is critical for ensuring faithful&#160;genome duplication.&#160;G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase &#948;, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.

This protocol outlines a fluorescence microscopy-based single-molecule DNA replication assay, enabling real-time visualization of interactions between DNA polymerases and obstacles such as G-quadruplex structures.