High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme

Abstract

Guanine-rich, single-stranded DNAs and RNAs that fold to G-quadruplexes (GQs) are able to complex tightly with heme and display strongly enhanced peroxidase activity. Phenolic compounds are particularly good substrates for these oxidative DNAzymes and ribozymes; we recently showed that the use of biotin-tyramide as substrate can lead to efficient GQ self-biotinylation. Such biotinylated GQs are amenable to polymerase chain reaction amplification and should be useful for a relatively non-perturbative investigation of GQs as well as GQ-heme complexes within living cells. Here, we report that in mixed solutions of GQ and duplex DNA in vitro, GQ biotinylation is specifically >104-fold that of the duplex, even in highly concentrated DNA gels; that a three-quartet GQ is tagged by up to four biotins, whose attachment occurs more or less uniformly along the GQ but doesn't extend significantly into a duplex appended to the GQ. This self-biotinylation can be modulated or even abolished in the presence of strong GQ ligands that compete with heme. Finally, we report strong evidence for the successful use of this methodology for labeling DNA and RNA within live, freshly dissected Drosophila larval salivary glands.

Figure 1.

Testing of self-biotinylation by various GQ-forming DNAs (A) and RNAs (B) as well as by a DNA (‘BLD’) and an RNA (‘r(ssRNA)’) that do not fold to GQ. Both panels A and B show 7.5% non-denaturing gels run in 50 mM TBE buffer at room temperature. ‘StAv’ refers to StAv, where it has been added subsequent to the standard biotinylation reaction (marked ‘+’) or where it has not been added (marked ‘−’). The percent values shown in a given ‘+’ lane refer to the percentage of DNA in that lane that has been mobility-shifted by virtue of complexation of the biotinylated DNA with the added StAv.

Figure 2.

How many biotins attach to each ‘CatG4’ GQ? (A) Design of the reference complex between a synthetic, singly biotinylated ‘CatG4’ DNA and its 1:1 complex with monoavidin(MAv). (B) Expected 1:1 complexation of monoavidin with biotin moieties attached to a ‘CatG4’ DNA via peroxidation of a BT substrate by the ‘CatG4’s’ oxidative complex formed with hemin. (C) Two exposures of a native polyacrylamide gel showing the appearance of up to four monoavidin-retarded bands (shown with red asterisks) formed from the reaction described in (B).

Figure 3.

The specificity of GQ biotinylation over ss or dsDNA in dilute co-solutions. (A) A schematic diagram for the experiment, in which co-dissolved 10⁴:1 molar ratios of ‘ssDNA’:‘CatG4-ext’ or of ‘dsDNA’:‘CatG4-ext’ are treated with hemin, H₂O₂ and biotin-tyramide (BT). (B and C) Native gels showing reciprocally ³²P-end-labeled ‘CatG4-ext’ (10 nM) co-dissolved 100 μM ‘ssDNA’ (B) or 100 μM ‘dsDNA’ (C). Upon treatment with StAv, retarded mobility (biotinylated) bands (indicated by red brackets) were observed and then quantitated relative to the unbiotinylated DNA in those same lanes.

Figure 4.

The specificity of GQ biotinylation over dsDNA in highly concentrated (gelated) dsDNA solutions. Co-dissolved 10⁴:1 molar mixtures of ‘dsDNA’:‘CatG4-ext’ were treated with hemin, H₂O₂ and biotin-tyramide (BT) either in dilute solution (‘-Salmon Sperm DNA’) or in a highly concentrated dsDNA solution (‘Salmon Sperm DNA: 17.5 mg/ml’)

Figure 5.

The distance range of biotinylation. (A) Top, the design of a duplex-GQ chimera, 46-nt ‘CatG4-T7’ hybridized to 22-nt ‘ssDNA’. Bottom, an 8% denaturing gel showing the individually ³²P-labeled component oligonucleotides, ‘CatG4-T7’ (band ‘1’) and ‘ssDNA’ (band ‘3’) that make up the duplex-GQ chimera. Bands ‘2’ and ‘4’ represent show, respectively, post-biotinylation ³²P-labeled ‘CatG4-T7’ (out of the duplex-GQ chimera containing non-radiolabeled ‘ssDNA’) and post-biotinylation ³²P-labeled ‘ssDNA’ (out of the duplex-GQ chimera containing non-radiolabeled ‘CatG4-T7’). The minor bands shown with a bracket (}) represent inter-strand crosslinked minor products formed between ‘ssDNA’ and ‘CatG4-T7’. (B) A native gel showing purified DNA isolated and purified from bands 1–4 shown in the denaturing gel (in A), run with either StAv added (+) or not added (−). The numbers shown in red indicate the percentage of total DNA StAv-shifted (and are therefore biotinylated) in the relevant lanes.

Figure 6.

Examination of biotinylation in an extended duplex linked to a GQ. (A) Does biotinylation extend past 31 bp in a duplex linked to a GQ? To the left are shown a GQ-duplex chimera consisting of three short oligonucleotides, ‘Comp-1’, ‘ssDNA’ and ‘Comp-3’, hybridized simultaneously to different stretches of the tailed GQ-forming oligonucleotide ‘CatG4-ext2’. Here, only either ‘CatG4-ext2’ or ‘ssDNA’ were 5′ ³²P-labeled (shown, respectively, as a blue asterisk and a green asterisk). The denaturing gel shows the radiolabeled ‘CatG4-ext2’ or ‘ssDNA’ from the complete GQ-duplex chimera, either biotinylated under the specified conditions or not. (B) Native gel showing StAv-shifted bands from DNA species 1–6 following purification from the denaturing gel shown in A. The bands shown with the red bracket are the StAv shifted bands. The multiple bands seen from ‘CatG4-ext2’ (shown with red arrows) represent different folded conformers formed in the native gel by this large oligomer. (C) The effective radius of biotinylation (∼10 nm, representing a duplex of ∼31 bp), estimated from the above experiments.

Figure 7.

The distribution of covalently appended biotins along the length of CatG4 DNA. (A) A schematic of the question being posed, about the distribution of biotin along the length of the CatG4 oligonucleotide. (B) The sequences of the four ‘CatG4’ variants, CatG4_Rx (where x = 1–3). The nucleotide marked in red is the single ribonucleotide within each of these oligonucleotides. The asterisk shows the site of ³²P-labeling (at the 5′ or the 3′ end). The numbers shown below each sequence indicate raw percentages of StAv-shifted bands relative to total DNA in a given gel band (such as shown in Supplementary Figure S2). The numbers obtained were from two independent sets of measurements. (C) Absolute percentages of the likelihood of biotin localization along segments of the total sequence of the ‘CatG4’ oligonucleotide.

Figure 8.

The competitive effect of GQ-binding ligands. (A) A schematic showing of 10 min treatments of ∼1 μM heme–GQ complex (made by complexation of 1 μM ‘CatG4-ext’ with 5 μM hemin) with 20 μM of, individually, NMM, pyridostatin and BRACO19 GQ-binding ligands. (B) A native gel showing StAv-retarded bands of biotinylated CatG4-ext complexed with added StAv (shown with a red arrow). The percentage of this retarded complex (with respect to the total DNA in a given lane) is indicated in blue above each lane.

Figure 9.

Biotinylation of RNA and DNA within live Drosophila melanogaster salivary glands. (A) A schematic diagram showing the design of the experiment. (B) Design of biotin dot-blots of total cellular RNA and genomic DNA. (C) Developed biotin dot blots from 2–5 μl (DNA = 200 ng; RNA = 400 ng for all dot blots) of total gland RNAs and genomic DNA extracted from live Drosophila larvae that have been treated with 50 μM heme, 3 mM BT and pulsed briefly (2–3 min) with 10 mM H₂O₂ followed by quenching. Blots ‘1’ show chemiluminescence from the full reaction, where all the above reagents are present; ‘2’, ‘3’ and ‘4’ show negative controls, where one of the participating reagents at a time is left out. The positive control spots, ‘Control Biotinyl-DNA’ show a standard 3′-biotinylated DNA spotted at two different concentrations: 2 ng (Spot 1) and 0.2 ng (Spot 2). (D) Developed biotin dot blots from 2–5 μl (DNA = 200 ng; RNA = 400 ng for all dot blots) of total cellular RNAs isolated from live salivary glands that have been treated with 50 μM heme plus 500 μM GQ-ligand (or 5 μM heme plus 250 μM GQ-ligand) and 3 mM BT, all pulsed briefly (2–3 min) with 10 mM H₂O₂ followed by quenching.