Fig 1.
Primer extension assay to evaluate presence of non-B DNA structure.
A. Schematic representation of the strategy of primer extension used for detecting pause sites due to altered DNA structures. Upon denaturation, the cloned sequence refolds into the non-B DNA (in this case a G-quadruplex) and acts as a replication block, preventing the extension of radiolabelled primer beyond the structure. This leads to the formation of a truncated product, which can be detected upon electrophoresis on a polyacrylamide gel. B. A denaturing polyacrylamide gel (8%) profile showing primer extension products using a radiolabelled primer SCR105 on increasing concentrations of the plasmid, pMN7 (0, 5, 10, 20, 50, 100 and 150 ng), containing the cloned wild type G-quadruplex forming motif, at an annealing temperature of 58°C. The pause site generated is marked with a square bracket. Lanes 1 and 2 are no DNA template controls without KCl and lane 3 is no DNA control with KCl. “M” is the radiolabeled 50 nt ladder and the molecular sizes are marked. In case of lanes 3–9, 10 mM KCl has been used. Wedge indicates the increasing concentration of pMN7.
Fig 2.
Primer extension assay using differentially positioned primers on a plasmid containing G-quadruplex forming motif.
A. Schematic representation for the use of multiple primers which bind at different distances from the G-quadruplex forming motif. As a result, based on the distance between the primer and the motif, different sized products are expected. B. Polyacrylamide gel profile for primer extension reactions using primers, SCR21, SCR105 and SCR83, on the plasmid, pMN7, at different annealing temperatures. The annealing temperatures used were 56, 58 and 60°C. The pause sites generated as a result of replication blocks are marked in square brackets. Lanes 1, 5 and 9 are no DNA template controls. “M” is the radiolabeled 50 nt ladder and the molecular sizes are marked. The distance between primers SCR21, SCR105 and SCR83 and the G-quadruplex forming region is 165, 111 and 67 nt, respectively.
Fig 3.
DMS protection assay to test G-quadruplex formation at BCL2 mbr.
A. Sequence of the G-rich strand at the BCL2 mbr. The stretches of guanines which are protected from DMS methylation are in red and marked by arrows, while the two guanines which react to DMS within the motif are marked by asterisks. B. The gel profile showing DMS sensitivity at G-quadruplex motif in the BCL2 mbr following chemical probing reaction. Lane 1, oligomeric DNA containing G-quadruplex motif treated with piperidine alone. Lane 2, DNA treated with DMS and piperidine following incubation in TE. Lane 3, DNA treated with DMS and piperidine after incubating in 100 mM KCl. C. A 2D model for the intramolecular G-quadruplex formed at the BCL2 mbr based on the reactivity of guanines to DMS. The guanines involved in the quartet formation are in red.
Fig 4.
Evaluation of G-quadruplex formation at BCL2 mbr, when present on a plasmid DNA.
A. Plasmid constructs, pMN7 and pMN8, were prepared by cloning the wild type and the mutant oligomeric sequence containing G-quadruplex motif from BCL2 mbr, respectively. B-C. CD spectra resulting from pMN7 and pMN8 in the absence (B) and presence (C) of KCl.
Fig 5.
Primer extension on wild type G-quadruplex motif and its mutant.
Primer extension using oligomer SCR105 on plasmids containing wild type (pMN7) or mutant (pMN8) G-quadruplex motif of BCL2 mbr. In each case, Lane 1 and 6 are primer alone, lanes 2, 3, 7, 8 are primer extensions in the absence of KCl and lanes 4, 5, 9, 10 are in the presence of 100 mM KCl. The pause sites are marked with a square bracket. “WT” denotes wild type and “MT” denotes mutant plasmids. Sequencing ladder was prepared using pMN7 with primer SCR105 by Sanger’s chain termination method of sequencing. A, C, G and T denotes the corresponding ddNTP-mediated chain termination reaction.
Fig 6.
Primer extension on wild type and mutant G-quadruplex motif at BCL2 mbr using multiple primers.
A-C. Primer extension using oligomers, SCR21 (A), SCR105 (B) and SCR83 (C) for wild type and mutant plasmids. In each case, lane 1 is primer alone, lanes 2–4 are primer extensions at different temperatures as indicated. The pause sites generated as a result of replication blocks due to G-quadruplex formation are marked with square brackets. “WT” denotes wild type and “MT” denotes mutant plasmids. “M” is 50 nt ladder.
Fig 7.
Evaluation of impact of different ions on G-quadruplex formation when BCL2 mbr is present on oligomeric DNA substrates.
A. Schematic representation of the oligomers used in the study. BTM1 (C) is the complementary sequence of G-quadruplex forming sequence and BTM2 (G) represents the G-quadruplex forming sequence of BCL2 mbr. BTM4 (G6) represents the mutant form of BTM2 (G) where 6 Gs are mutated (indicated in blue color). B-E. Different oligomeric DNA, BTM1, BTM2 and BTM4 were incubated either in TE buffer (B), TE + KCl (C), TE + NaCl (D) or TE + LiCl (E) at 37°C for 1 h and resolved on a 12% native PAGE at room temperature. In all cases both the gel running buffer (TBE) and gel contained respective ions (100 mM). Formation of intermolecular species are marked by square brackets, while intramolecular G-quadruplexes and substrate DNA are indicated by arrows.
Fig 8.
Primer extension through wild type and mutant G-quadruplex motif in the presence of different ions.
A. Primer extension using oligomer SCR105 on pMN7 plasmid in the presence of increasing concentrations of KCl, (0, 5, 10, 20, 50, 75, 100 and 125 mM), which is indicated using a wedge. Lane 1 is no DNA control. B, C. Primer extension using oligomer SCR105 for wild type (B) and mutant (C) plasmids in the presence of either KCl, LiCl, NaCl or CaCl2 (100 mM). In panel B, lane 1 is no DNA control. The pause sites generated as a result of replication block due to G-quadruplex formation are marked by a square bracket. “M” is 50 nt ladder.