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

G-quadruplexes are nucleic acid secondary structures that are 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 G4 within potential G-quadruplex-forming sequences is a biologically relevant issue, which is currently addressed primarily through computational tools.

To support such predictions and detect 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-quadruplexes. The key advantage by B-CePs is that it reduced the number of steps in the protocol and it reduced 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. Begin by pipetting G4-TBA single-stranded TBA and double-stranded TBA solutions into three different tubes for folding.

Heat the samples to 95 C for five minutes. Let them cool slowly to room temperature. Once the tubes have cooled to room temperature, centrifuge the tubes.

Next, organize three sets each of seven empty autoclaved 0.5 mL tubes. Add 3 L of ultrapure water into each tube. Pipette 5 L of the folded nucleic acid constructs into each tube of the set.

Next, add 2 L of either 25 or 250 M B-CeP 1 to obtain the final probe concentration of 5 and 50 M respectively. To the three control tubes, add ultrapure water instead of the compound and incubate all the samples at 37 C.Stop the reaction by placing the tubes at 20 C.Then dry the samples in a vacuum centrifuge. Begin by preparing a polymerized PAGE gel sandwich.

Remove the clamps and paper tape layers and slowly remove the comb. Thoroughly rinse the wells with distilled water. Place the gel sandwich correctly in the vertical electrophoresis apparatus.

Fill the top and bottom reservoirs with TBE buffer. Warm up the plates by performing a pre-run of the gel electrophoresis for at least 30 minutes at 50 Watts. Meanwhile, resuspend the dried samples in 5 L of denaturing gel-loading buffer.

Next, use a small syringe filled with TBE buffer to flush the urea out of the wells of the gel. Now load the samples into the clean wells, taking note of the odor of loading. Run the gel for two hours at 50 Watts or until the bromophenol blue dye has run down two thirds of the gel.

After electrophoresis, turn off the power supply, carefully remove the glass sandwich, and clean the glass plates. Place the gel in a gel imager to detect the fluorescence of the FAM labeled oligonucleotide bands. After one, four and 15 hours of incubation, either 5 or 50 M B-CeP 1 reacted with 2 M samples of G4-TBA, single-stranded TBA and double-stranded TBA.

Controls were loaded for each set of samples. TBA substrate showed only one alkylation adduct due to the availability of only G8 for alkylation and subsequent strand cleavage. Multiple adducts and bands of cleavage products were observed in single and double stranded TBA because all guanines were susceptible to B-CeP reaction.

Probing reaction and Tris Buffer resulted in inefficient chemical mapping due to the presence of undesired nucleophiles, causing reduced probe reactivity. The G4-TBA samples showed only the formation of adducts without stranded cleavage. For the single and double stranded TBA, only a 24-hour incubation led to DNA fragmentation comparable to the 15-hour fragmentation without Tris.