7168 Nucleic Acids Research, 2011, Vol. 39, No. 16 Figure 2. Equilibrium binding GMSAs of puriﬁed recombinant G4R1/RHAU incubated with unimolecular G4-DNA-containing oligonucleotides yield apparent Kd values in the low picomolar range. (A, B, E, F, I and J) are representative (of three repetitions) phosphorimages of non-denaturing gel electropherograms of GMSAs; the top triangles indicate the direction of increasing G4R1/RHAU concentration. GMSA of 1 pM 50-[32P]-labeled (A) unimolecular Poly A Zic1 47-mer G4-DNA, (E) unimolecular Poly T Zic1 47-mer G4-DNA, (I) c-Myc 51-mer G4-DNA incubated with a broad range of concentrations of G4R1/RHAU (lane 1, 0 pM; lane 2, 1 pM; lane 3, 5 pM; lane 4, 10 pM; lane 5, 20 pM; lane 6, 40 pM; lane 7, 80 pM). GMSA of 1 pM 50-[32P]-labeled unimolecular (B) Poly A Zic 47-mer G4-DNA, (F) Poly T Zic1 47-mer G4-DNA, (J) c-Myc 51-mer G4-DNA incubated with concentrations of G4R1/RHAU that were increased in small (0.5–1 pM) increments between (B) lanes 1–12, (F) lanes 1–10, or (J) lanes 1–6, followed by larger increments up to 80 pM. Binding data for Poly A Zic1 47-mer G4-DNA, Poly T Zic1 47-mer G4-DNA, and c-Myc 51-mer G4-DNA were directly ﬁt by non-linear regression to a hyperbolic equation (Panels C, G and K, respectively) and to a double reciprocal linear equation by linear regression (Panels D, H and L, respectively). Error bars are Mean ± SD, n = 3 independent experiments. pM of Poly T Zic1 G4-DNA 47-mer DNA bound the enzyme (Figure 2E, lane 4). The bound Poly T Zic1 G4-DNA-enzyme complex appeared as a main single band with a faster electrophoretic mobility than the Poly A Zic1 G4-DNA 47-mer-enzyme complex (compare Figure 2A and E). In addition to the main band of Poly T Zic1 G4-DNA:G4R1/RHAU complex, slower mobility molecular complexes were also observed in the upper most regions of the gel. Careful titration of the G4R1/RHAU concentration in the binding reactions (Figure 2F) demonstrated a Kd for the Poly T Zic1 G4-DNA:G4R1/ RHAU interaction of 2.4 ± 0.2 pM (Figure 2G and H). Binding of the G4-DNA-containing c-Myc 51-mer of complete native sequence (without 30- or 50-unstructured sequence tags) to G4R1/RHAU was also evaluated by GMSA. Similar to the Zic1 G4-DNA oligonucleotides, >50% of 1 pM c-Myc G4-DNA 51-mer was shifted in the presence of 10 pM G4R1/RHAU (Figure 2I, lane 3). The bound complexes appeared as two main band regions in the gel, with the most prominent complex observed with the slowest mobility band near the loading origin of the gel, suggesting possible self-association of G4-DNA: G4R1/RHAU bound complexes. Careful titration of the G4R1/RHAU concentration in the binding reactions (Figure 2J) demonstrated a Kd for the c-Myc 51-mer G4-DNA:G4R1/RHAU interaction of 4.4 ± 0.6 pM (Figure 2K and L). Overall, GMSA results indicate the enzyme possesses a remarkably tight and similar dissoci- ation constant for each of the G4-DNA-containing oligonucleotides. In order to determine whether tight binding of G4R1/ RHAU is speciﬁc for oligodeoxyribonucleotides that contain G4-DNA structures, we measured apparent binding afﬁnity of the enzyme for a number of 50-[32P]- end-labeled control oligonucleotides (Figure 3A–D), each of which did not contain G4-DNA structure by the CD