7170 Nucleic Acids Research, 2011, Vol. 39, No. 16 criteria discussed above (Figure 1I and J and data not shown). In all cases, G4R1/RHAU concentrations were titrated up to 300 pM. In most cases there was little or no detectable binding. Minimal binding of the scrambled Zic1 oligonucleotide by G4R1/RHAU was observed in Figure 3B (lanes 4–7); however, the percentage of DNA oligonucleotide bound never approached 50% (the con- centration for an apparent Kd), even in the presence of 300 pM enzyme. Therefore, it is clear that if G4R1/ RHAU binds any of these non-G4-DNA-containing oligonucleotides, the apparent Kd for the bound complex is more than 50-fold higher than the apparent Kd’s observed for G4R1/RHAU-unimolecular G4-DNA complexes. The question remained, however, whether the tight binding interaction of G4R1/RHAU with G4-DNA sequence-containing oligonucleotides was based solely on recognition and binding of G4-DNA structure, or would a guanine-rich sequence, with or without G4-DNA struc- ture, sufﬁce to interact tightly with G4R1/RHAU? In order to address this question, the 33-mer DNA oligo- nucleotide Z33, which contains runs of d(pG) (Table 1) that self-anneal into a parallel stranded tetramolecular G4 structure (36), was incubated with increasing concentra- tions of G4R1/RHAU in either an unstructured single-stranded form, or in tetramolecular G4-DNA form. Oligonucleotide Z33 did not bind G4R1/RHAU when present in unstructured single stranded form (Figure 3E, lanes 1–7). In contrast, G4-DNA-formed Z33 tightly bound G4R1/RHAU, even in the presence of molar excess of unstructured single-stranded Z33 (Figure 3E, lanes 8–14). An additional test was performed to conﬁrm that the G4R1/RHAU binding interaction with d(pG) run- containing DNA oligonucleotides requires G4-DNA structure. Poly A Zic1 DNA 47-mer was treated with di- methyl sulfate in order to methylate guanine N7, thereby preventing guanine quartet formation by Hoogsteen bonding. Dimethyl sulfate treated Poly A Zic1 DNA 47-mer did not bind G4R1/RHAU (Supplementary Figure S3). Taken together, these data indicate that tight G4R1/RHAU binding of oligonucleotides containing d(pG) runs requires the presence of G4-DNA structure, not simply a guanine-rich sequence. Finally, it was determined whether the low Kd of the G4-DNA:G4R1/RHAU binding interaction was a result of slow release of bound G4-DNA, as is often the case for tight protein:ligand interactions. Release of bound Poly A Zic1 47-mer G4-DNA from a complex with G4R1/RHAU was determined by incubating a complex containing 1 pM 50-[32P]-labeled Poly A Zic1 G4-DNA bound to 200 pM G4R1/RHAU at 37 C in the presence of 20 nM unlabeled Poly A Zic1 G4-DNA . This is a 20 000-fold molar excess of unlabeled to labeled G4-DNA and a 100-fold molar excess of unlabeled G4-DNA to enzyme. Figure 3F, lane 2 shows that this added cold blocking material prevents G4R1/RHAU from appreciably binding [32P]-labeled G4-DNA. A nonlinear regression analysis of loss of binding with time produced a t1/2 of binding of 67 ± 9 h. These data indicate a koff of 2.9 10 6/s. Given that the GMSA data displayed in Figure 2 indicates a Kd for the Poly A Zic1 G4-DNA:G4R1/RHAU interaction of 5 pM, the predicted kon is 5.2 105/M/s is reasonable for a diffusion based event. This experiment independently conﬁrms the tight binding of unimolecular G4-DNA by G4R1/RHAU in a manner independent of quantiﬁcation of the concentration of G4R1/RHAU enzyme. Hybridization of a complementary PNA to the Poly A Zic1 DNA 47-mer forms a complex that does not contain G4-DNA structure and is not bound tightly by G4R1/RHAU The assertion that G4R1/RHAU binds directly to G4- DNA structure within unimolecular DNA oligonucleo- tides was further tested by speciﬁcally inhibiting the formation of the G4 structure with minimal disruption of the overall base accessibility of the oligonucleotide. Base pair hybridization with a Watson–Crick complementary PNA was used to inhibit G4-DNA formation of the Poly A Zic1 DNA 47-mer. PNAs are synthetic DNA mimics for which N-(2-aminoethyl) glycine moieties replace the sugar phosphate backbone of DNA. In a PNA the bases are arranged on a backbone that typically has no negative charge. Therefore, PNAs bind and base pair to target se- quences with signiﬁcantly greater stability than cognate DNA oligonucleotides, and unlike DNA:DNA duplexes, the stability of PNA:DNA duplexes remains high in low salt conditions. Previously, PNAs have been experimentally utilized for trapping putative G4-forming DNA oligo- nucleotides in a non-G4 state (45,46). A 12-mer PNA sequence, 50-NH2-CCCCCCCCACCC- lysine that is complementary to positions 11–23 d(pGGGT GGGGGGGG) on the Poly A Zic1 DNA 47-mer sequence was tested for its ability to inhibit the formation of G4-DNA by the Poly A Zic1 DNA oligonucleotide. Figure 4C, lane 1 shows the mobility following a boiling and cooling cycle of monomeric Poly A Zic1 DNA 47-mer upon non-denaturing gel electrophoresis. The addition of PNA at a 10:1 PNA:DNA molar ratio prior to a boiling and cooling cycle (Figure 4C, lane 2) caused the appear- ance of two new bands of slower electrophoretic mobility. CD spectroscopy of these two new bands which were puriﬁed electrophoretically (Supplementary Figures S4 and S5), as well as stoichiometric analysis (data not shown) indicated that the lower of the two new bands (denoted as Band 2 in Supplementary Figures S4 and S5) is a unimolecular form of Poly A Zic1 47-mer that may be a structural isomer of unimolecular Poly A Zic1 G4-DNA stabilized by the presence of the PNA, while the upper band (denoted as Band 3 in Supplementary Figures S4 and S5) is a PNA:DNA hybrid that does not possess G4 structure. Band 2 ex- hibited stabilization by increasing KCl concentration (Supplementary Figure S4B), which is characteristic of G4-DNA. The lack of G4 structure and the presence of the PNA:DNA hybrid was conﬁrmed by its CD spectral behavior under no salt conditions; the molar ellipticity at 263 nm of the PNA:DNA hybrid was not destabilized by increasing temperature in the no salt condition, as expected for a PNA:DNA hybrid that maintains stability under low salts conditions (Supplementary Figure S5,