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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Nucleic acid and chemical probe preparation

1. Nucleic acids
   NOTE: The oligonucleotide named "TBA" is the 15-mer DNA sequence 5'-GGT-TGG-TGT-GGT-TGG-3' labeled at the 3'-end by the fluorophore 5-carboxyfluorescein (FAM) to enable visualization on the gel. The unlabeled oligonucleotide "cTBA" is its DNA complementary sequence 5'-CCA-ACC-ACA-CCA-ACC-3'. TBA and cTBA are employed to obtain the three different structures, as shown in Table 1

Discussion

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.......

Materials

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