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</str...

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/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>

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.

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

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplex are nucleic acid secondary structures that typically formed within guanine-rich DNA sequences. The chemical mapping of G4 by B-CePs aims at the in vitro characterization of G4, and allows the identification of the specific guanines forming the G-tetrads. The identification and the experimental validation of G4s within potential G-quadruplex-forming sequences is a biologically relevant issue, which is currently addressed primary through computational tools.To support such predictions and detect the unpredicted G4, chemical mapping by B-CePs represents an accessible method to identify G4 in DNA sequences. The chemical mapping by B-CePs is a convenient alternative to the widely used dimethyl sulfate footprinting protocol for the characterization of G-quadruplex. The key advantage by B-CePs is that it reduces the number of steps in the protocol and it reduces the initial amount of the DNA substrate.The chemical mapping by B-CePs is described here with the TBA sequence as a proof of concept. The protocol will lead to new experiments applicable to any other potential G-quadruplex-forming sequence exploring both DNA and RNA constructs.

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

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

in-vitro-chemical-mapping-g-quadruplex-dna-structures-bis-3

Research

JoVE Journal

Biochemistry

826 ビュー.  University of Padova. G-四重鎖は、通常、グアニンに富むDNA配列内に形成される核酸二次構造です。B-CePによるG4の化学マッピングは、G4のin vitro特性評価を目的としており、G-tetradsを形成する特定のグアニンの同定を可能にします。潜在的なG-四重鎖形成配列内のG4の同定と実験的検証は、生物学的に関連する問題であり、現在、計算ツールによって主に対処されています。このような予?...