G-Quadruplex formation using fluorescent oligonucleotide as a detection method for discriminating AGG trinucleotide repeats

Transcription

1 Electronic Supplementary Material (ESI) for ChemComm. This journal is The Royal Society of Chemistry 1 Electronic Supplementary Information G-Quadruplex formation using fluorescent oligonucleotide as a detection method for discriminating AGG trinucleotide repeats Yoojin Park, Ki Tae Kim and Byeang Hyean Kim* Section Experimental Methods Table S1. MALDI-TOF mass spectral data Figure S1. Fluorescence emission spectra of PYGs Figure S. UV Vis spectra of PYGs Figure S3. Fluorescence emission spectra of Figure S. UV Vis spectra of PYGs in the presence of DA3A and RA3A Figure S5. Fluorescence emission spectra of with targets induced to form secondary structure Figure S. UV melting curves Figure S7. Fluorescence emission spectra of, C, A, and G Figure S8. CD spectra of Figure S9. CD spectra of with target sequence DA3A and RA3A Figure S1. Fluorescence Job s plot analysis of with DA3A and RA3A Figure S11. CD spectra of in the presence of the target sequences at various ratios Figure S1. Fluorescence emission spectra of with DA and RA Figure S13. CD spectra of with target sequence DA and RA Figure S1. Fluorescence Job s plot analysis of with DA and RA Figure S15. Fluorescence intensity of in the presence of DA and RA at various concentrations Figure S1. Fluorescence intensity of in the presence of AGG repeats at various concentrations Figure S17. Fluorescence intensity added to an intramolecular G-quadruplexes Figure S18. CD spectra of in the presence of with DT3T, DC3T, RU3U, and RC3C Figure S19. Relative fluorescence responses of with variety of G-quadruplex-forming sequences Refernces

2 Experimental Methods Oligonucleotide synthesis Natural oligonucleotides were obtained commercially from Integrated DNA Tehnologies (IDT). Modified oligonucleotides were synthesized on a CPG support (scale: 1 μmol; pore size: 1 Å) using standard phosphoramidite methods on an automated DNA synthesizer (POLYGENE DNA synthesizer). The synthesized oligonucleotides were cleaved from the solid support upon treatment with 3% aqueous NH OH (1. ml) for 1 h at 55 C. The crude products from the automated ODN synthesis were lyophilized and diluted with distilled water (1. ml). The ODNs were purified through reversed-phase HPLC (Merck LichoCART C18 column; 1 5 nm; 1 µm; pore size: 1 Å). The HPLC mobile phase was held isocratically for 1 min with 5% acetonitrile/.1 M triethylammonium acetate (TEAA) ph 7. buffer at a flow rate of.5 ml/min; the gradient was then increased linearly over 1 min from 5 to 5 % acetonitrile/.1 M TEAA at the same flow rate. The fractions containing the purified ODNs were cooled and lyophilized. 8% aqueous AcOH was added to the ODNs. After 1 h at ambient temperature, the AcOH was evaporated under reduced pressure. The residue was diluted with water (1. ml); this solution was then purified through HPLC using the same conditions as those described above. The ODNs were analyzed using reversed-phase HPLC. The products were characterized using MALDI-TOF mass spectrometry. Oligonucleotide sample preparation for fluorescence and UV spectroscopy In a 1.5 ml microtube, a 15 μm target oligonucleotide sample (1 μl) was added to M KCl (5 μl) and voltexmixed for.5 min. A PYG probe (1 equiv.) was added to the mixture. After mm Tris-HCl buffer (ph 7., μl) and water were added to give a total volume of 1 ml, the solution was voltex-mixed for 3 min, providing the sample for fluorescence measurement. For annealed species, the sample was held at 9 C for 3 min and then cooled under at ambient conditions for 3 h. Fluorescence emission spectra of the oligonucleotides were measured with excitation at 385 nm using a quartz cuvette (path length: 1 cm) and a Varian Carry Eclipse spectrometer. UV spectra were recorded using a Cary 1 Conc UV Vis spectrometer (Varian) and a quartz cell (path length: 1 cm). Circular dichroism (CD) spectroscopy CD spectra of oligonucleotides were recorded using a JASCO J-81 spectropolarimeter equipped with a temperature controller. For each sample, 1 spectral scans were accumulated at C over wavelengths in the range from to 3 nm. Samples of each oligonucleotide were prepared at a concentration of. μm in 1 mm Tris-HCl buffer and 5 mm KCl.

3 Table S1 MALDI-TOF mass spectral data Sequence Calcd. (m/z) Found (m/z)) PYG PYG PYG A C G PYG3

4 PYG PYG5 A

5 C G

6 (A) (C) PYG3 PYG3+DA3A PYG3+RA3A PYG5 PYG5+DA3A PYG5+RA3A PYG PYG+DA3A PYG+RA3A Figure S1. Fluorescence emission spectra of (A) PYG3, PYG, and (C) PYG5 with the target sequences DA3A and RA3A in the presence of 5 mm K + ions. (1.5 μm PYG; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml; excitation wavelength: 385 nm)

7 (A) (C) (D) Figure S. UV Vis spectra, displaying the pyrene absorbance region of (A), PYG3, (C) PYG, and (D) PYG5 constructs in the absence and in the presence of 5 mm KCl. (1.5 μm PYG; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml) (A) Fluorescence intensity(a.u.) DA3A +DA3A+KCl RA3A +RA3A+KCl Figure S3. Fluorescence emission spectra of with the target sequences (A) DA3A and RA3A, recorded in the absence and presence of 5 mm K + ions (1.5 μm ; 1 mm Tris-HCl buffer, ph 7.; total volume of

8 sample: 1 ml; excitation wavelength: 385 nm) (A).8 +DA3A +RA3A.8 PYG3 PYG3+DA3A PYG3+RA3A (C) (D).8 PYG PYG+DA3A PYG+RA3A.8 PYG5 PYG5+DA3A PYG5+RA3A Figure S. UV Vis spectra, displaying the pyrene absorbance region of (a), (b) PYG3, (c) PYG, and (d) PYG5 constructs with the target sequences DA3A and RA3A, recorded in the presence of 5 mm K + ions. (1.5 μm samples; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml), DA3A+ RA3A+ Figure S5. Fluorescence emission spectra of in the presence of target sequence, DA3A and RA3A, which induce to form a secondary structure (1.5 μm ; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume

9 of sample: 1 ml; excitation wavelength: 385 nm) (A) RA3A (C) (E) Temperature ( ) 8 1 Temperature ( ) RA3A DA3A (D) Temperature ( ) Temperature ( ) +DA3A Temperature ( ) Figure S. UV melting curves of (A), and RA3A, (C) RA3A, (D) and DA3A, and (E) DA3A at 95 nm. Their melting temperatures were recorded at 58.7 in and RA3A, and 5. in RA3A, respectively. (5. µm of samples; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml)

10 (A) Fluorescence intensiy (a.u.) C A G +DA3A C+DA3A A+DA3A G+DA3A Fluorescence intensiy (a.u.) RA3A C+RA3A A+RA3A G+RA3A Figure S7. Fluorescence emission spectra of, C, A, and G (A) alone and with the target sequence DA3A and with the target sequence RA3A, recorded in the presence of 5 mm KCl, after annealing (. μm PYG; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml; excitation wavelength: 385 nm) +KCl Figure S8. CD spectra of, recorded in the presence and absence of K + ions, after annealing (total oligonucleotide concentration. μm; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml, C)

11 (A) DA3A+5 mm KCl +DA3A DA3A+5 mm KCl DA3A RA3A+5 mm KCl +RA3A RA3A+5mM KCl RA3A Figure S9. CD spectra of with (A) DA3A and RA3A recorded in the absence and presence of 5 mm KCl, after annealing (oligonucleotide concentration:. μm; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml, C) (A) Fl. Int. at 8 nm (a.u.) Inflection point at []/([]+[DA3A]) Fl. Int. at 8 nm (a.u.) 3 1 Inflection point at []/([]+[RA3A]) Figure S1. The fluorescence Job s plot analysis at 8 nm of with the target sequences (A) DA3A and RA3A (total concentration. μm; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml; excitation wavelength: 385 nm) Job s plot analysis was performed by systematic variation of the molar fraction of and individual target sequences while keeping a constant total concentration μm for interaction with DA3A, RA3A. The mixture of and G-quadruplex-forming sequences were prepared as above, and the fluorescence emission intensity at 8 nm were recorded.

12 (A) DA3A (3. :.8) +DA3A (. : 1.) +DA3A (. :.) +DA3A (1. :.) +DA3A (.8 : 3.) DA3A 1 8 (.) +RA3A (3. :.8) +RA3A (. : 1.) +RA3A (. :.) +RA3A (1. :.) +RA3A (.8 : 3.) RA3A (.) Figure S11. CD spectra of with the target sequences (A) DA3A and RA3A at different ratios of probe to target sequence, after annealing (total oligonucleotide concentration:. μm; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml; C) (A) DA +RA DA +RA Figure S1. Fluorescence emission spectra of with the target sequences DA and RA, recorded in the presence of 5 mm KCl, (A) without and with annealing (1.5 μm ; target sequence; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml; excitation wavelength: 385 nm)

13 (A) DA +DA3A DA RA +RA3A RA Figure S13. CD spectra of with (A) DA and RA recorded in the presence of 5 mm KCl, after annealing (oligonucleotide concentration:. μm; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml, C) (A) Fl.Int. at 8 nm (a.u.) 8 Inflection point at []/([]+[DA]) Fl. Int. at 8 nm (a.u.) 1 Inflection point at []/([]+[RA]) Figure S1. The fluorescence Job s plot analysis at 8 nm of with the target sequences (A) DA and RA (total concentration. μm; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml; excitation wavelength: 385 nm)

14 (A) (C) 1 8 Fl. Int. at 8 nm (a.u.) Concentration (μm) (D) Fl. Int. at 5 nm (a.u.) Concentration (μm) Fl. Int. at 8 nm (a.u.) Concentration (μm) Fl. Int. at 5 nm (a.u.) Concentration (μm) Figure S15. Fluorescence intensities of, recorded in the presence of (A) DA and RA at various concentrations, without annealing and (C) DA and (D) RA at various concentrations with annealing (.5 μm of ; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml; excitation wavelength: 385 nm) (A) Concentration of AGG repeat (μm) DA DA8 DA Concentration of AGG repeat (μm) RA RA8 RA1

15 Figure S1. Fluorescence intensity of in the presence of (A)DNA and RNA AGG repeats at various concentrations and lengths without annealing (.5 μm of ; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml; excitation wavelength: 385 nm) DA : 3 -d(agg) DA8 : 3 -d(agg) 8 DA1 : 3 -d(agg) 1 RA : 3 -r(agg) RA8: 3 -r(agg) 8 RA1: 3 -r(agg) DA+ RA+ Figure S17. Fluorescence intensities after had been added to the intramolecular G-quadruplexes of DA and RA. Before measuring the fluorescence intensity, the samples of DA and RA with 5 mm KCl had been incubated for days at C to allow enough time to form the G-quadruplexes (1.5 µm ; 1 mm Tris-HCl buffer, ph 7.; total volume of sample: 1 ml; excitation wavelength: 385 nm)

16 (A) 1 1 DT3T+ DT3T DT DC3C+ DC3C DC (C) (D) 1 8 RU3U+ RU3U RU RC3C+ RC3C RC Figure S18. CD spectra of with the target sequences (A) DT3T, DC3C, (C)RU3U, and (D) RC3C, recorded in the presence of K + ions, at different ratios of probe to target sequence, after annealing (total oligonucleotide concentration:. μm; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl; total volume of sample: 1 ml; C). DT : d(tgg TGG TGG TGG) RU : r(ugg ugg ugg ugg) DC : d(cgg CGG CGG CGG) RC : r(cgg cgg cgg cgg)

17 DA RA pu18 c-kit vegf bcl c-myc htelo (F-F)/F RA DA pu18 c-kit vegf bcl hteloc-myc Figure S19. (A) Fluorescence enhancement upon formation of (3+1) intermolecular G-quadruplexes in presence of a variety G-quadruplex-forming sequence and relative fluorescence intensities. (F : Fluorescence emission intensity of, 1.5 μm ; 1 mm Tris-HCl buffer, ph 7.; 5 mm KCl, total volume of sample: 1 ml; excitation wavelength: 385 nm) c-myc 5 - TGA GGG TGG GGA GGG TGG GGA A vegf 5 - GGG CGG GCC GGG GGC GGG c-kit 5 - GGG CGG GCG CGA GGG AGG GG bcl 5 - GGG CGC GGG AGG AAT TGG GCG GG pu AGG GTG GGG AGG GTG GGG htelo 5 -A(GGGTTA) 3 GGGT 1.