7172 Nucleic Acids Research, 2011, Vol. 39, No. 16 G4-DNA structure) at 37 C in 30 min and allow signiﬁ- cant PNA hybridization, as is accomplished by long term incubation of the Poly A Zic1 DNA 47-mer with PNA at 55 C or by heating (95 C) and cooling cycles in high concentrations of PNA (Figure 4C, lane 3). Observation of a PNA:DNA hybrid in the presence of enzyme would indicate that, not only does G4R1/RHAU bind G4-DNA-containing oligonucleotides, but the enzyme opens the unimolecular G4 structure as well. Our PNA trap assay was largely modeled upon a previous study that assayed the kinetics of the folding and unfolding of telomere DNA oligonucleotide in the presence of a com- plementary PNA trap (45). Kinetic considerations (7) indicate that if G4R1/RHAU unwound Poly A Zic1 DNA 47-mer G4-DNA structure under physiological con- ditions and then released the unstructured product, the oligonucleotide would likely snap back within seconds into a G4-DNA structure, producing the futile cycle as diagramed in Figure 5A. However, if a molar excess of base-complementary PNA were present during the Figure 5. A G4R1/RHAU resolution assay of Poly A Zic1 DNA 47-mer G4-DNA in the presence of a complementary PNA shows that G4R1/ RHAU unwinds the G4-DNA structure. (A) Schematic diagram illustrates the kinetic futile cycle problem of attempting to determine if G4R1/ RHAU unwinds the G4-DNA structure in the PQS-containing oligonucleotide. (B) Schematic diagram illustrating how the kinetic futile cycle problem is solved with a PNA trap assay. (C) Phosphorimage of representative (of three repetitions) nondenaturing gel electropherogram of 100 pM 50-[32P]-labeled Poly A Zic1 DNA 47-mer incubated with increasing concentrations of a PNA with Watson–Crick complementarity to the G4-DNA-forming sequence in the absence of ATP and G4R1/RHAU (lanes 1–4), with 10 mM ATP plus increasing concentrations of G4R1/RHAU, but without PNA (lanes 5–8), with ATP and PNA plus increasing concentrations of G4R1/RHAU that had been pre-heated to 95 C for 5 min (lanes 9–14), or with ATP and PNA plus increasing concentrations of active G4R1/RHAU (lanes 15–20). (D) Phosphorimage of representative (of three repetitions) nondenaturing gel electropherogram of 1 nM 50-[32P]-labeled Poly A Zic1 DNA 47-mer incubated with PNA and ATP plus increasing concentrations of a G4R1/RHAU mutant that lacks ATPase activity (lanes 1–6), with PNA plus increasing concentrations of wild-type G4R1/ RHAU, but without ATP (lanes 7–12), or with PNA, ATP, and increasing concentrations of wild type G4R1/RHAU (lanes 13–18); lanes 19–21 are a titration of 1 nM 50-[32P]-labeled Poly A Zic1 DNA 47-mer incubated with increasing concentrations of PNA in the absence of G4R1/RHAU and presence of 10 mM ATP.