showed greater stabilization by K+ (Figure 1H), although its Tm in both 50 mM KCl and LiCl was somewhat lower than the Poly A Zic1 DNA oligonucleotide in the presence of the corresponding cation. The CD spectrum of the scrambled Zic1 47-mer control (which has the same nucleotide content but a different sequence order as the Poly A Zic1 DNA 47-mer) exhibited an ellipticity peak at 263 nm; however, this signal was completely lost by raising the temperature to 40 C. Furthermore, the peak of ellipticity at 263 nm observed in the scrambled Zic1 47-mer spectrum diminished by 50% at a lower tem- perature in KCl than in LiCl (Figure 1J), a behavior which is inconsistent with G4-DNA structure. Therefore, these CD results indicate that G4-DNA was formed in the Poly A Zic1 DNA 47-mer oligonucleotide and the c-Myc 51-mer oligonucleotide, but not in the scrambled Zic1 47-mer control DNA. The temperature and cation dependence of the CD spectra of a number of other oligonucleotides that were used in G4R1/RHAU binding studies (Figure 3) were similarly assessed (data not shown). In all cases, only single-stranded DNA oligonucleotides with canonical G4-DNA sequences conforming to the unimolecular G4-DNA formulae (14,15) met the criteria of: (i) exhibit- ing a positive peak of ellipticity at 263 nm; (ii) displaying a CD spectrum that was stable above 37 C but which destabilized at near 95 C; and (iii) exhibiting a 50% decrease in signal of ellipticity at 263 nm (deﬁned as a Tm) at a signiﬁcantly higher temperature in 50 mM KCl- than in 50 mM LiCl-containing buffer. Oligonucleotides that failed to meet these criteria and also failed to exhibit a positive peak of ellipticity at 290 nm were inter- preted as not forming true unimolecular G4-DNA struc- ture. All putative non-G4-DNA-forming control sequences used in these studies exhibited CD spectra that failed to meet these 3-fold criteria for the presence of G4-DNA structure. Although the CD spectra and cation-dependent thermal stability studies indicate that the Poly A Zic1 DNA 47-mer and c-Myc 51-mer oligonucleotides anneal into par- allel stranded G4-DNA structures, these studies do not distinguish intramolecular from intermolecular G4-DNA. In this regard, stoichiometry studies were per- formed to determine whether the G4-DNA structure formed by Poly A Zic1 DNA 47-mer was intra- or inter- molecular. A 1 nM Poly A Zic1 DNA 47-mer was mixed with equimolar poly A Zic 1 DNA 37-mer prior to forming G4-DNA by our standard annealing protocol. Poly A Zic1 DNA 37-mer has an identical G4-DNA- forming sequence to Poly A Zic1 DNA 47-mer; the only difference between these two oligonucleotides is that the core sequence in Poly A Zic1 DNA 37-mer is ﬂanked only on the 50-end by d(pA)10, whereas Poly A Zic1 DNA 47-mer is ﬂanked on both ends. Non- denaturing PAGE after annealing the mixed oligonucleo- tides revealed only bands whose electrophoretic mobility corresponded to that of Poly A Zic1 37-mer G4-DNA and Poly A Zic1 47-mer G4-DNA (Supplementary Figure S1). No bands of intermediate electrophoretic mobility indica- tive of intermolecular G4-DNA structure formation between oligonucleotides of different lengths were Nucleic Acids Research, 2011, Vol. 39, No. 16 7167 observed. Thus, under our annealing conditions, poly A Zic DNA forms an intramolecular G4-DNA structure exclusively. G4R1/RHAU binds unimolecular G4-DNA with an apparent Kd in the picomolar range while non-G4- containing DNA does not display tight binding The ability of G4R1/RHAU to bind unimolecular G4-DNA was ascertained by the gel mobility shift assay (GMSA). In previous studies, we observed that the addition of EDTA to the G4R1/RHAU resolution buffer inhibits unwinding of, but not binding to, tetramolecular G4-nucleic acid structures, allowing us to determine an apparent Kd for binding tetramolecular G4-DNA (37). This same approach was used to determine apparent Kd’s for unimolecular G4-DNA-containing oligonucleotides and non-G4-DNA-containing control oligonucleotides. Binding reactions were performed by incubating increasing concentrations of recombinant full-length human G4R1/RHAU enzyme with a ﬁxed con- centration of 50-[32P]- end-labeled oligonucleotides. A ﬁne titration of increasing concentrations of the DNA oligo- nucleotide Poly A Zic1 DNA 47-mer with a ﬁxed concen- tration of G4R1/RHAU indicated that 70% of G4R1/ RHAU molecules possessed DNA binding activity (Supplementary Figure S2; a single site binding model was assumed). Figure 2A, E and I shows representative GMSAs of three DNA oligonucleotides (Poly A Zic1 47-mer DNA, Poly T Zic1 47-mer DNA and c-Myc 51-mer DNA), each of which possess unimolecular G4-DNA by CD criteria; each of these oligonucleotides binds G4R1/RHAU at remarkably low enzyme concentrations. We initially studied binding of G4R1/RHAU to an oligonucleotide (termed Poly A Zic1 DNA 47-mer) with d(pA)10 tails attached to both the 50 and 30of a 27-mer sequence present in the 50 untranslated region of the coding sequence of the transcription factor Zic1 (Table 1). The d(pA)10 tails were added as unstructured ends that might maximize performance of the enzyme. Nearly 80% of the 1 pM Poly A Zic1 G4-DNA 47-mer was bound at a G4R1/RHAU concentration of 10 pM (Figure 2A, lane 4). The bound complexes formed a broad band of electrophoretic mobility in the higher molecular weight region of the gel, demonstrating retarded migration. In order to determine the apparent Kd for the Poly A Zic1 G4-DNA:G4R1/RHAU inter- action, a ﬁne titration was performed and the concentra- tion of G4R1/RHAU was raised in 0.5–1 pM increments in the binding reactions (Figure 2B). The percent bound Poly A Zic1 G4-DNA as a function of G4R1/RHAU con- centration was ﬁt directly using Equation (1) (Figure 2C) and via a double reciprocal plot (Figure 2D) to a hyberbolic, saturable, single site binding model. Kd for the Poly A Zic1 DNA:G4R1/RHAU interaction was 5.7 ± 0.2 pM. To determine if the d(pA)10 sequence inﬂuenced the low apparent Kd observed, we replaced the d(pA)10 tails with d(pT)10 tails of the same length and repeated the GMSA. At a concentration of 10 pM of G4R1/RHAU >80% of 1