the amount of enzyme that can unfold 0.2 pmol of tetramolecular Z33 quadruplex in 30 min at 37 C (36). Addition of increasing amounts of unlabeled unimolecular G4-DNA-containing Poly A Zic1 DNA 47-mer (Figure 6A, lanes 3–12), c-Myc 51-mer (Figure 6C, lanes 3–12), or tetramolecular pZ33 G4-DNA (Figure 6E, lanes 3–12) caused a competitor concentration-dependent decrease in the rate of G4R1/RHAU-catalyzed tetramolecular G4-DNA resolution. The c-Myc 51-mer unimolecular G4-DNA quadruplex inhibited the enzymatic resolution of tetramolecular G4-DNA substrate at lower inhibi- tor:substrate molar ratios than did the Poly A Zic DNA 47-mer (compare Figure 6C to A). The data were analyzed graphically as the percent of maximal activity (([activity measured with competitor at given molar ratio]/[activity measured without competi- tor]) 100) as a function of the unlabeled competi- tor:labeled substrate molar ratio (Figure 6B, D and F). We have previously used this assay to demonstrate that 300-fold molar excesses of Y form DNA, 50 overhang duplex DNA and 30 overhang duplex DNA were required to inhibit resolution of 50-TAMRA-labeled tetramolecular Z33 G4-DNA as effectively as unlabeled tetramolecular G4-DNA (36). As expected, addition of unlabeled tetra- molecular pZ33 G4-DNA inhibited resolution of 50-TAMRA-labeled tetramolecular Z33 G4-DNA by 50% at a 1:1 molar ratio of unlabeled:labeled DNA (Figure 6F). Poly A Zic1 47-mer DNA caused 50% inhib- ition of G4R1/RHAU-catalyzed resolution of tetra- molecular G4-DNA at a 6:1 competitor:substrate molar ratio (see dotted line at abscissa intercept in Figure 6B), indicating that it is a much better inhibitor than Y form DNA and duplex DNA from previous studies (36). However, the c-Myc 51-mer caused 50% inhibition at a 0.06:1 unlabeled competitor:labeled substrate molar ratio, indicating that it inhibits resolution of tetramolecular G4-DNA 100-fold better than the Poly A Zic1 DNA 47-mer G4-DNA. DISCUSSION Previously our laboratory identiﬁed G4R1/RHAU as the protein responsible for the majority of HeLa cell tetramolecular G4-DNA resolving activity by using a highly stringent afﬁnity chromatography approach select- ive for tetramolecular G4-DNA binding proteins (36). Selection for speciﬁcity was achieved by utilizing over a 100-fold ratio of blocking single-stranded d(pHHN)11 DNA to target tetramolecular G4-DNA (attached to paramagnetic beads) (36). d(pHHN)11 DNA was chosen as a non-speciﬁc blocking DNA because the sequence could not form intramolecular or intermolecular G4-DNA structures. We expected the isolated protein would bind tightly to tetramolecular G4-DNA. Indeed, recombinant G4R1/RHAU was shown to bind tightly to tetramolecular G4-DNA with a Kd of 77 ± 6 pM (37). However, it remained unknown whether G4R1/RHAU could bind and unwind unimolecular G4-DNA. Although tetramolecular G4-DNA and unimolecular G4-DNA share many structural commonalities, there are Nucleic Acids Research, 2011, Vol. 39, No. 16 7175 differences between the structures, such as DNA looping found in unimolecular G4-DNA structures, which could present steric interference to enzymatic recognition. In addition, the issue of whether G4R1/RHAU recognizes the unimolecular G4-DNA structure addresses the poten- tial physiologic role(s) of this enzyme because the forma- tion of unimolecular G4-DNA has a low kinetic barrier of formation under intracellular conditions, and unimolecu- lar G4-DNA is the most common G4-DNA structure expected to form in vivo. Therefore, the current study was essential to determine whether G4R1/RHAU could speciﬁcally bind to unimolecular G4-DNA and open that structure. The results presented here indicate that G4R1/RHAU binds unimolecular G4-DNA with remarkable tightness (Kd of 5.7 ± 0.2 pM for Poly A Zic1 47-mer and 4.4 ± 0.6 pM for c-Myc). These estimations of Kd have at least one fundamental caveat: the accuracy of the esti- mates of Kd is dependent upon accurate protein quantiﬁ- cation through estimating Coomassie staining of G4R1/ RHAU protein against standards of known protein quantities on an SDS gel. Comparison of mass estimation of puriﬁed proteins, e.g. bovine serum albumin or E. coli b-galactosidase, by ultraviolet light absorption spectros- copy with estimates obtained by Coomasie staining of these proteins in SDS gels indicate that the two methods differ by 20%–49% (<2-fold, data not shown). Therefore, the Kd estimate reported here could be off by as much as 2-fold due to the method of estimation of protein content. Additionally, the modest free enzyme concentrations required for measuring these low Kd’s can be diminished due to signiﬁcant amounts of protein being bound to DNA during binding reactions, thus limiting the precision of the Kd determinations to upper boundary estimates in these cases. However, despite these qualiﬁcations, the con- clusion stands that G4R1/RHAU binds G4-DNA with remarkably tight binding afﬁnity. In addition, a determin- ation of the t1/2 of binding at 67 ± 7 h made in this study is protein concentration independent and further afﬁrms the low Kd and tight binding results found for G4R1/RHAU interaction with unimolecular G4-DNA. To our knowledge, these are the lowest Kd’s reported for a protein binding to G4-DNA. The next lowest Kd’s in the literature for protein binding to G4-DNA is 77 pM for G4R1/RHAU binding to tetramolecular DNA (37) and 79 pM for the mouse otholog mXRN1p of the yeast Xrn1 or Kem1 protein binding with tetramolecular DNA (47). The afﬁnity found between G4R1/RHAU and unimolecular G4-DNA is tighter than that found in most antigen- antibody binding interactions (48). The G4R1/RHAU unimolecular G4-DNA binding interaction has substantial ligand speciﬁcity, considering that control DNA sequences not possessing G4-DNA structure bind G4R1/RHAU with at least a 50-fold poorer magnitude of afﬁnity. Even DNA oligonucleotides with G-rich regions capable of forming quadruplex DNA but not participating in a G4-DNA structure, were not bound tightly by G4R1/ RHAU. Furthermore, inhibition of G4-DNA formation within the PQS of Poly A Zic1 DNA 47-mer by a PNA 12-mer whose base sequence was complementary to the PQS of the oligonucleotide abolished the tight afﬁnity