resolution reaction, the PNA could hybridize and trap the opened structure oligonucleotide as a PNA:DNA hybrid (Figure 5B). Non-denaturing gel electrophoretic analysis of the en- zymatic PNA:DNA hybrid trap assay demonstrated G4R1/RHAU-catalyzed unwinding of Poly A Zic1 DNA 47-mer G4-DNA (Figure 5C). Total of 100 pM Poly A Zic1 DNA oligonucleotide was incubated at 98 C with 0, 10, 100 nM, or 10 mM of complementary PNA, respectively. After a 10 min incubation the material was cooled to room temperature, then analyzed electro- phoretically (Figure 5C, lanes 1–4). In the absence of PNA, two bands that represent two structural isomers of Poly A Zic1 DNA 47-mer were observed (Figure 5C, lane 1). In the presence of 10–100 nM complementary PNA, the slower mobility G4-DNA containing structural isomer of Poly A Zic1 DNA 47-mer is present, but not the faster mobility isomer (Figure 5C, lanes 2 and 3) sug- gesting that the PNA stabilizes one structural isomer of Poly A Zic1 DNA 47-mer without forming a stable PNA:DNA hybrid. Increasing amounts of a third slower mobility band was observed as the PNA concentration increased from 10 nm to 10 mM (Figure 5C, lanes 2–4). CD of a puriﬁed band of similar mobility suggested this band was a PNA:DNA hybrid (Supplementary Figure S5). Nearly all of the Poly A Zic1 DNA 47-mer was found in the PNA:DNA hybrid band in the presence of 10 mM PNA (Figure 5C, lane 4); there was also a smear of even slower mobility species observed in the presence of 10 mM PNA (Figure 5C, lane 4). We speculate that this smear represents other less stable PNA:DNA hybrids. A small amount of a slower migrating band (present at approximately half way down the gel) was also observed in all lanes of the electropherogram except when catalytically active G4R1/RHAU and ATP were present (Figure 5C, lanes 7 and 8, 18–20); it is possible that this band represents a multimolecular Poly a Zic1 G4-DNA that is resolved by active G4R1/RHAU. When Poly A Zic1 G4-DNA 47-mer was incubated in the presence of G4R1/RHAU in resolution buffer con- taining 100 mM KCl and 10 mM ATP, but without com- plementary PNA, only substrate Poly A Zic1 47-mer G4-DNA (principally faster migrating G4-DNA isomer, with a very small amount of slower mobility G4-DNA isomer) and increasing amounts of slow mobility G4R1/ RHAU-oligonucleotide complexes were observed as the enzyme concentration increased (Figure 5C. lanes 5–8). This result was similar to the GMSAs of G4R1/RHAU incubated with PolyA Zic1 G4-DNA in the presence of EDTA discussed earlier (compare Figure 5C, lanes 5–8 with Figure 2A). The addition of 10 nM complementary PNA under identical conditions changed the products observed after G4R1/RHAU-Poly A Zic1 G4-DNA 47-mer incubation (Figure 5C, lanes 15–20). In the pres- ence of complementary PNA, but without G4R1/RHAU, Poly A Zic1 DNA 47-mer only adopted a conformation with the electrophoretic mobility of the upper G4-DNA band (compare Figure 5C, lanes 9 and 15). However, as increasing amounts of G4R1/RHAU were titrated into the reaction (Figure 5C, lanes 16–20), the amount of the G4-DNA band decreased, while the PNA:DNA duplex Nucleic Acids Research, 2011, Vol. 39, No. 16 7173 band increased in intensity. This observation suggests that the complementary PNA hybridizes and traps the DNA oligonucleotide in a PNA:DNA hybrid once the G4-DNA structure has been resolved by G4R1/RHAU. In order to assess whether formation of the PNA:DNA hybrid was dependent upon the catalytic activity of intact G4R1/RHAU, the Poly A Zic1 47-mer containing G4-DNA was incubated with complementary PNA in the presence of increasing concentrations of boiled (denatured) G4R1/RHAU. In these reactions there was no increase in the appearance of the PNA:DNA duplex (Figure 5C, lanes 10–14), although a slow mobility smear was observed at the highest G4R1/RHAU concentration (Figure 5C, lane 14), suggesting the boiled enzyme was still capable of some binding of DNA, albeit not as efﬁciently as the native enzyme (Figure 5C, lanes 5–8). These results indicate that G4R1/RHAU catalytically opens the G4 structure of the Poly A Zic1 DNA 47-mer, allowing the PNA to hybridize to the complementary sequence in the DNA oligonucleotide; denatured G4R1/RHAU cannot open the G4-DNA structure to allow PNA/DNA duplex formation. In order to further demonstrate that unwinding of Poly A Zic1 DNA 47-mer with formation of a PNA:DNA duplex in the presence of G4R1/RHAU required catalytic activity of G4R1/RHAU, resolution reactions were carried out in which wild-type G4R1/RHAU was replaced by G4R1/RHAU containing a E!A substitution mutation at amino acid position 335 in the Walker B box. The ATPase activity of G4R1/RHAU is abolished by this mutation (40). No PNA:DNA duplex band was observed after incubation with mutant G4R1/RHAU with 1 nM G4-DNA (Figure 5D, lanes 2–6), although slow mobility bound mutant G4R1/RHAU:G4-DNA complexes were observed. In contrast, PNA:DNA duplex formation was observed after incubation with wild type G4R1/RHAU at concentrations identical to those of mutant G4R1/RHAU (Figure 5D, lanes 14–18). PNA:DNA duplex formation in the presence of wild-type G4R1/RHAU was not observed if ATP was absent from the reaction (Figure 5D, lanes 8–12), or if 10 mM AMP-PNP, the non-hydrolyable ATP analog, was substi- tuted for ATP (Supplementary Figure S7). These data conﬁrm that formation of PNA:DNA duplex in the pres- ence of G4R1/RHAU, indicative of G4-DNA unwinding, requires G4R1/RHAU ATPase activity. Competition studies indicate that unimolecular G4-DNA containing-oligonucleotides inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA; the c-Myc oligonucleotide is an effective competitor at competitor:substrate molar ratios <<1 Inhibition of the resolution of 50-TAMRA-labeled tetramolecular G4-DNA Z33 oligonucleotide into single- stranded DNA was assessed by titrating increasing molar ratios of unlabeled unimolecular G4-DNA-containing oligonucleotides into resolution reactions consisting of a ﬁxed amount of G4R1/RHAU and 2 nM 50-TAMRA- labeled tetramolecular G4-DNA substrate (Figure 6). Upon completion of the resolution reaction, the extent