Self-biotinylation of DNA G-quadruplexes via intrinsic peroxidase activity

Abstract

The striking and ubiquitous in vitro affinity between hemin and DNA/RNA G-quadruplexes raises the intriguing possibility of its relevance to biology. To date, no satisfactory experimental framework has been reported for investigating such a possibility. Complexation by G-quadruplexes leads to activation of the bound hemin toward catalysis of 1- and 2-electron oxidative reactions, with phenolic compounds being particularly outstanding substrates. We report here a strategy for exploiting that intrinsic peroxidase activity of hemin•G-quadruplex complexes for self-biotinylation of their G-quadruplex component. Such self-biotinylation occurs with good efficiency and high discrimination in vitro, being specific for G-quadruplexes and not for duplexes. The biotinylated DNA, moreover, remains amenable to polymerase chain reaction amplification, rendering it suitable for analysis by ChIP-Seq and related methods. We anticipate that this self-biotinylation methodology will also serve as a sensitive tool, orthogonal to existing ones, for identifying, labeling and pulling down cellular RNA and DNA G-quadruplexes in general, as well as proteins bound to or proximal to such quadruplexes.

Figure 1.

Schematic for covalent biotinylation of G-quadruplexes via the intrinsic peroxidase activity of G-quadruplex•heme complexes.

Figure 2.

(A) Self-biotinylation of G-quadruplex DNA (G4). Lane 1: Control (5 μM hemin; 1 μM DNA; 60 μM streptavidin added following 30 min reaction). Lane 2: 5 μM hemin; 1 μm DNA; 5 μM bio-tyr; 60 μM streptavidin added after 30 min. Lane 3: 5 μM hemin; 1 μM DNA; 50 μM bio-tyr; 500 μM H₂O₂; 60 μM streptavidin added after 30 min. (B) Self-biotinylation of G-quadruplex DNA. Determination of the key components for successful bio-tyr reaction-mediated G4 biotinylation.

Figure 3.

Hemin-dependence, biotin-tyramide dependence and time dependence for G4 biotinylation. (Upper gel) The default condition was 30 min reactions of 1 μM CatG4, 1 μM hemin, 5 μM bio-tyr and 250 μM H₂O₂ (for bio-tyr dependence, [bio-tyr] = 0–25 μM (plotted in the middle graph); for hemin dependence, [hemin] = 0–5 μM (plotted in the lowest graph). (Lower gel) the reaction contained 1 μM CatG4, 1 μM hemin, 5 μM bio-tyr and 250 μM H₂O₂. Time aliquots were quenched by the addition of catalase enzyme to degrade residual H₂O₂. These data are plotted in the top graph. All data report the mean values obtained from two independent experiments. The error bar indicates one standard deviation from the mean. The asterisks show streptavidin-shifted G4 bands.

Figure 4.

Investigation of whether bio-tyr-mediated covalent biotinylation of G4 DNA enables such DNA to be amplified by PCR. (A) A scheme for treatment of G4 with bio-tyr to generate biotinylated DNAs and also various unbiotinylated controls. In block (1) G4 ‘stock’ refers to G4 DNA that has not been subjected to a bio-tyr-mediated biotinylation reaction. Block (2) ‘bulk reaction’ refers to G4 DNA that has subjected to the bio-tyr reaction, but not subsequently subjected to a purification protocol to separate out biotinylated from unbiotinylated DNA. shows ‘L’ and ‘H’ refer to low (20 μM) and high (200 μM) concentrations of bio-tyr, respectively. (B) A further elaboration on blocks (3) and (4). In this preparatory gel, lane 1 shows bio-tyr reaction conditions but excluding hemin; lane 2 shows the reaction excluding bio-tyr; lane 3 excludes H₂O₂; lane 4 shows the full reaction with all components, including 20 μM bio-tyr; lane 5 shows full reaction but with 200 μM bio-tyr. (C) Gel showing the results of experiment schematized in A and B. The thick arrow to the right of the gel indicates the lightly ³²P-labeled template (84 nt) whereas the star to the right indicates the PCR-amplified product (74 nt). ‘P’ refers to the ³²P-labeled reverse primer. ‘L’ shows a reference ladder. The arrow on the left shows the 5′ end of the guanine-rich motif that forms the G-quadruplex.