Involvement of G-triplex and G-hairpin in the multi-pathway folding of human telomeric G-quadruplex.

DNA constructs

All oligonucleotides required to make the DNA substrates were purchased from Sangon Biotech (Shanghai, China). Sequences and labeling positions of all the oligonucleotides were listed in Supplementary Table S1 . For DNA constructs used in smFRET measurements, DNA was annealed with 1:3 mixture of the stem and G4 or ssDNA strands by incubating the mixture at 95°C for 5 min, then slowly cooling down to room temperature in about 7 h. The strand without biotin was used in excess to reduce the possibility of having non-annealed strand anchored at the coverslip surface. The concentration of stem strand was 2.5 μM and all annealing were carried out in annealing buffer containing 20 mM Tris–HCl, pH 8.0 with different concentrations of NaCl or KCl. Buffers Buffers containing 20 mM Tris–HCl, pH 8.0 with different concentrations of NaCl or KCl were used. For single-molecule FRET measurements, 0.8% D-glucose, 1 mg/ml glucose oxidase (266600 units/g, Sigma), 0.4 mg/ml catalase (2000–5000 units/mg, Sigma) and 4 mM Trolox were added to prevent the photobleaching and photoblinking. Single-molecule fluorescence data acquisition Single-molecule FRET study was carried out with a home-built objective-type total-internal-reflection microscopy. Cy3 was excited by 532 nm Sapphire laser (Coherent Inc., USA). An oil immersion objective (100 ×, N.A.1.49) was used to generate an evanescent field of illumination. Fluorescence signal from Cy3 and Cy5 were split by a dichroic mirror, and finally collected by an electron-multiplying charge-coupled device camera (iXON, Andor Technology, South Windsor, CT, USA). The coverslips (Fisher Scientific, USA) and slides were cleaned thoroughly by a mixture of sulfuric acid and hydrogen peroxide, acetone and sodium ethoxide, then the surfaces of coverslip were coated with a mixture of 99% mPEG (m-PEG-5000, Laysan Bio, Inc.) and 1% of biotin-PEG (biotin-PEG-5000, Laysan Bio, Inc.). Streptavidin (10 μg/ml) were added to the microfluidic chamber made of the PEG coated coverslip, and incubated for 10 min. After washing, 50 pM DNA were added to the chamber and allowed to be immobilized for 10 min. Then free DNA was removed by washing with the imaging buffer (20 mM Tris–HCl, pH 8.0, different concentrations of NaCl or KCl, 0.8% D-glucose, 1 mg/ml glucose oxidase, 0.4 mg/ml catalase and 4 mM Trolox). We used an exposure time of 100 ms for all single-molecule measurements at a constant temperature of 22°C. FRET data analyses The FRET efficiency was calculated using I A /(I D +I A ), where I D and I A represent the intensity of donor and acceptor respectively. The FRET value above 1 is due to background subtraction from very low intensity in the donor channel, giving rise to negative donor intensity. Basic data analysis was carried out by scripts written in Matlab, and all data fitting were generated by Origin 8.0. Single-molecule FRET histograms were generated by picking the initial 50–100 frames of each trace from ∼300 molecules, and fitted by multi-peak Guassian distributions, with the peak position unconstrained. Transition density plots (TDP) ( 48 ) were constructed from smFRET traces showing dynamic transitions using free available Hidden Markov Model (HMM) and TDP software from Professor Taekjip Ha’s website ( http://bio.physics.illinois.edu/HaMMy.asp ). Circular dichroism spectropolarimetry CD experiments were performed with a Bio-Logic MOS450 / AF-CD optical system (BioLogic Science Instruments, France) equipped with a temperature controlled cell holder, using a quartz cell with 1-mm path length. A 2 μM solution of DNA was prepared in 20 mM Tris–HCl, pH 8.0 with different cation. The solution was incubated at 95°C for 5 min, then slowly cooled down to room temperature in about 7 h. CD spectra were recorded in the UV (220–320 nm) regions in 0.75 nm increments with an averaging time of 2 s at 25°C.

Gel electrophoresis assay

A total of 12 nt poly-T sequence was labeled by FAM fluorophore at 3′ end for visualization (T 12 -FAM, Supplementary Table S1 ). DNA for gel assay was annealed with 1:1 mixture of T 12 -FAM and 3G2 or 4G2 sequences by incubating the mixture at 95°C for 5 min in 100 mM NaCl or KCl, then slowly cooling down to room temperature in about 7 h. Afterward both T 12 -FAM and annealing products were subjected to 15% native PAGE (polyacrylamide gel electrophoresis), run at room temperature and visualized by Gel imaging analysis system (Gel Doc-It310, UVP, USA).

MD simulations

MD simulations and available volume (AV) simulations were performed to calculate the FRET efficiencies for several DNA constructs ( 25 , 49 ). To get the initial conformations for AV simulations, we carried out all-atom MD simulations for anti-parallel G4, hybrid-2 G4, 2-tetrad anti-parallel G4, G-triplex and G-hairpin using GROMACS5.0.6 with AMBER99SB-ILDN force field ( 50 ). The solvent (water model: TIP3P) and 100 mM KCl were added to build a charge-neutral system. We chose KCl as G4 was known to be more stable when the central ion is K + compared with Na + ( 17 ). All systems were energy-minimized and equilibrated successfully. MD simulations were performed at 1 bar and 298 K with Parrinello-Rahman pressure coupling ( 51 ) and Velocity-rescaling temperature coupling schemes ( 52 ). For each system, the simulations were run for 100 ns according to previous studies ( 25 , 44 ). The constructs of anti-parallel G4 (2MCO.pdb), hybrid-2 G4 (2JSL.pdb) and 2-tetrad anti-parallel G4 (2KF8.pdb) were taken from RCSB Protein Data Bank. The anti-parallel G4 structure in 2MCO is highly consistent with 143D which has been chosen by several earlier MD simulation studies ( 44 , 53 ). G-triplex and G-hairpin were constructed from the mutation of anti-parallel G4 according to the sequences in Supplementary Table S1 . Relevant nucleotides of anti-parallel G4 were replaced with dTs, and energy-minimization was run for 100 ps before further MD simulations. The double-stranded DNA was built in Nucleic Acid Builder. Available volume simulations The AV simulations were performed to calculate the FRET efficiency for each system ( 49 ). Frames for AV simulations were extracted from the MD simulation trajectories by every 10 ps, implying that there are 10 000 frames for each of the five systems. The parameters for Cy3/Cy5 dye pairs are listed in Supplementary Table S2 . During AV simulations, the atom O3′ of the last nucleotide in the G4 constructs was selected as the attachment point for Cy3 and the methyl group carbon atom C7 of T6 in the complementary stand was selected as the attachment point for Cy5. Hydrogen atoms which were attached to the O3′ and C7 atoms were deleted before AV simulations. The Förster radius was set as 60 Å ( 47 ).

Supplementary Material Supplementary Data Click here for additional data file.