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

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	<li>Davis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quartets+40+years+later:+from+5'-GMP+to+molecular+biology+and+supramolecular+chemistry.">G-quartets 40 years later: from 5'-GMP to molecular biology and supramolecular chemistry.</a> Angewandte Chemie. 43 (6), 668-698 (2004).</li><li>Varshney, D., Spiegel, J., Zyner, K., Tannahill, D., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+and+functions+of+DNA+and+RNA+G-quadruplexes.">The regulation and functions of DNA and RNA G-quadruplexes.</a> Nature Reviews Molecular Cell Biology. 21 (8), 459-474 (2020).</li><li>Chambers, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+sequencing+of+DNA+G-quadruplex+structures+in+the+human+genome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+a+bis-3-chloropiperidine+derivative+incorporating+an+anthraquinone+pharmacophore.">Synthesis and evaluation of a bis-3-chloropiperidine derivative incorporating an anthraquinone pharmacophore.</a> Bioorganic &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bis-3-chloropiperidines+containing+bridging+lysine+linkers:+Influence+of+side+chain+structure+on+DNA+alkylating+activity.">Bis-3-chloropiperidines containing bridging lysine linkers: Influence of side chain structure on DNA alkylating activity.</a> Bioorganic &amp; Medicinal Chemistry. 23 (6), 1241-1250 (2015).</li><li>Zuravka, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+DNA+cleavage+activity+of+bis-3-chloropiperidines+as+alkylating+agents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-CePs+as+cross-linking+probes+for+the+investigation+of+RNA+higher-order+structure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bis-3-chloropiperidines+targeting+TAR+RNA+as+a+novel+strategy+to+impair+the+HIV-1+nucleocapsid+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+bis-3-chloropiperidines+as+RNA+modulators+targeting+TAR+and+TAR-protein+interaction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+topoisomerase+II+mediated+DNA+damage+by+bis-3-chloropiperidines:+The+importance+of+being+an+earnest+G.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+single-stranded+DNA+molecules+that+bind+and+inhibit+human+thrombin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-stranded+DNA+aptamer-binding+site+of+human+thrombin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behind+the+mirror:+chirality+tunes+the+reactivity+and+cytotoxicity+of+chloropiperidines+as+potential+anticancer+agents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appended+aromatic+moieties+in+flexible+bis-3-chloropiperidines+confer+tropism+against+pancreatic+cancer+cells.">Appended aromatic moieties in flexible bis-3-chloropiperidines confer tropism against pancreatic cancer cells.</a> ChemMedChem. 16 (5), 860-868 (2021).</li><li>Kypr, J., Kejnovska, I., Renciuk, D., Vorlickova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+dichroism+and+conformational+polymorphism+of+DNA.">Circular dichroism and conformational polymorphism of DNA.</a> Nucleic Acids Research. 37 (6), 1713-1725 (2009).</li><li>Onel, B., Wu, G., Sun, D., Lin, C., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+mobility+shift+assay+and+dimethyl+sulfate+footprinting+for+characterization+of+G-quadruplexes+and+G-quadruplex-protein+complexes.">Electrophoretic mobility shift assay and dimethyl sulfate footprinting for characterization of G-quadruplexes and G-quadruplex-protein complexes.</a> Methods in Molecular Biology. 2035, 201-222 (2019).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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/ 
			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

JoVE Journal

Biochemistry

1.2K 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 ...

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 ...

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 ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...