7164 Nucleic Acids Research, 2011, Vol. 39, No. 16 Moreover, G4R1/RHAU catalyzes unwinding of unimol- ecular G4-DNA into an unstructured form capable of hy- bridization with a Watson–Crick complementary peptide nucleic acid (PNA). MATERIALS AND METHODS Circular dichroism spectropolarimetry Circular dichroism (CD) experiments were performed on an AVIV Model 202 CD spectrometer equipped with a thermoelectrically controlled cell holder. A 1 mM solutions of G4-DNA were prepared in 10 mM Tris–HCl, pH 7.5, 1 mM EDTA ± 0–50 mM KCl or LiCl. Quartz cells with 1 cm path length were used for all experiments. CD spectra were recorded in the UV (200–350 nm) regions with 1 nm increments with an averaging time of 2 s. Thermal stability of G4-DNA was assessed by recording molar ellipticity at 263 nm as a function of temperature. The temperature was raised in 2 C increments; stirred samples were allowed to equilibrate for each temperature setting for 30 s. Molar ellipticity data were collected in the temperature range 25–98 C. G4-DNA formation and quantiﬁcation Oligonucleotides [unlabeled or 50 5,6-carboxytetramethyl- rhodamine (TAMRA)-labeled] were purchased from either Oligos Etc. or Integrated DNA Technologies. Tetramolecular G4-DNA was formed from oligonucleo- tide Z33 (Table 1) by a previously described procedure (33) that results in over 99% conversion of monomer to tetramolecular G4-DNA. Unimolecular G4-DNA was formed in various oligonucleotides (Table 1) by dissolving them at a concentration of 0.5 mM in 10 mm Tris–HCl, 1 mM EDTA, pH 7.5. Oligonucleotide solutions were ali- quoted into polymerase chain reaction (PCR) tubes and incubated in a thermocycler (Eppendorf epGradient S) at 98 C for 10 min, then held at 80 C. Immediately the tubes were opened and KCl was added to a ﬁnal concentration of 25 mM. The tubes were reclosed and allowed to come slowly to room temperature. Aliquots were combined and stored at 4 C for 2–3 days. Tetramolecular G4-nucleic acids formed from 50-TAMRA-labeled oligonucleotides were further puriﬁed by electrophoresis on a 10% poly- acrylamide gel and band excision. Electrophoretic bands containing G4-DNA were recovered by electroelution in a Schleicher and Schuell Elutrap with 1 TBE (89 mM Tris-Borate, pH 8.3, 2.5 mM EDTA) buffer additionally Table 1. DNA oligonucleotides used in this work containing 3 mM KCl. The Elutrap was run with buffer recirculation. 50-TAMRA-labeled G4 nucleic acids were aliquoted and stored at 20 C or lower. G4-DNA concentrations were determined by absorb- ance of 260 nm light using a Smart Spec 3000 UV spec- trophotometer (Bio-Rad). The molar extinction coefﬁcient for each oligonucleotide was determined by the instru- mental software after inputting the base composition of each. 50-[32P]-end labeling of G4 nucleic acids [32P]-end-labeled G4 oligonucleotides were obtained by incubating unlabeled oligonucleotides previously treated to form G4-DNA as described above, with T4 polynucleo- tide kinase (Promega Corporation) and [g-32P] ATP for 0.5 h at 37 C, according to the manufacturer’s instruc- tions. 50-[32P] end-labeled oligonucleotides were puriﬁed with a MicroSpin G25 column (GE Healthcare) equili- brated with TEK (10 mM Tris, 1 mM EDTA and 50 mM KCl) buffer and stored at 20 C. Gel mobility shift assay and apparent Kd determination for G4R1/RHAU-oligonucleotide complexes We have previously described the isolation of recombinant G4R1/RHAU from Rosetta 2 cells (36, 37). It should be noted that we have found that the addition of 0.02% b-lactalbumin protein as a stabilizer is important for iso- lation of enzyme of high-speciﬁc activity. However, because b-lactalbumin carrier is the dominant protein in preparations of G4R1, it was necessary to quantify G4R1 with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie blue staining instead of using solution quantiﬁcation tech- niques. Puriﬁed recombinant G4R1/RHAU was quantiﬁed in the following protocol: 15 ml of puriﬁed recombinant G4R1/RHAU was loaded on a standard 5% stacking/ 12% resolving SDS–polyacrylamide gel and the loading was compared to known amounts of standard; 0.0625, 0.125, 0.250, 0.500 and 0.750 mg of Promega Broad Range Molecular weight markers per band (cat no V849A). The gels were stained with ProtoBlue Safe Colloidal Coomassie G-250 stain (National Diagnostics, EC-722) per manufacturer’s instructions, destained and scanned on an Epson Perfection 2450 Photo scanner with Epson SilverFast TWAIN. Densitometric measurements of bands representing recombinant G4R1/RHAU and three proteins in the marker that were of similar size Oligo name Poly A Zic1 DNA (47-mer) c-Myc DNA (51-mer) Scrambled Zic1 DNA (47-mer) Poly T Zic1 DNA (47-mer) (HHN)11 DNA (33-mer) PolyA DNA (47-mer) Poly T DNA (47-mer) PNA (11-mer) Z33 DNA (33-mer) Sequence (50–30) 50-AAA AAA AAA AGG GT GGG GGG GCG GGG GAG GCC GGG GAA AAA AAA AA-30 50-GGC CGC TTA TGG GGA GGG TGG GGA GGG TGG GGA AGG TGG GGA GGA GAC TCA-30 50-AGA AGA GAG AGA GTG AGA GAG ACG AGA GAG GCC GAG AAG AGA GAG AG-30 50-TTT TTT TTT TGG GTG GGG GGG GCG GGG GAG GCC GGG GTT TTT TTT TT-30 50-HHN HHN HHN HHN HHN HHN HHN HHN HHN HHN HHN-30 50-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA AA-30 50-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TT-30 H2N-CCC CCC CCA CCC-Lys-COOH 50-AAA GTG ATG GTG GTG GGG GAA GGA TTT TCG AAC-30