Detection of cofilin mRNA by hybridization-sensitive double-stranded fluorescent probes

We have developed hybridization-sensitive fluorescent oligonucleotide probes that, in the presence of quencher strands, undergo efficient fluorescence quenching through the formation of partial DNA/DNA duplexes. In the presence of target RNA, rapid displacement of the quencher strands results in highly enhanced fluorescence.


Contents S1
Experimental details S2  Table S1. Probe sequences, containing Py U units, for each target sequence S3 Figure S1. UV absorption spectra of P1-P6 in the absence and presence of the target RNA S4 Figure S2. Fluorescence emission spectra of P4-P6 in the absence and presence of the target T21 S5 Table S2. Fluorescence enhancements of the probe sequences for the target RNA S6 Figure S3. Melting curves of P1-P3 in the presence of T19 and P4-P6 in the presence of T21 S7 Table S3. Melting temperature of each probe with its target RNA S8  Table S4. Quencher sequences, containing a Py U or Dab U unit, partially complementary to P1 S9 Figure S4. Structure of the internal quencher Dab U S10 Figure S5. UV absorption spectra and normalized UV absorption spectra of P1 in the presence of U5-U7 S11 Figure S6. Temperature-dependent absorption spectra of single-stranded P1 and P1 in the presence of a quencher strand U5-U6 S12 Figure S7. Fluorescence emission spectra of single-stranded P1 and U5-U7 and P1 in the presence of a quencher strand U5-U7 and T19 S13 Figure S8. UV absorption spectra of P1 in the presence of Q5-Q7 S14 Figure S9. Fluorescence emission spectra of P1 in the presence of Q5-Q7 and T19 S15 Figure S10. Melting curves of P1 in the presence of U5-U7 and Q5-Q7 S16 Table S5. Melting temperature of P1 in the presence of U5-U7 and Q5-Q7 S17 Figure S11. CD spectra of P1 and T19 and P1 in the presence of a quencher strand U5-U7 and T19 S18 Figure S12. Native polyacrylamide gel electrophoresis (PAGE) images of P1 with T19 in the presence of U5-U7 S19 Figure S13. Time-dependent fluorescence intensity of P1 in the presence of U7 after the addition of T19

Synthesis of oligonucleotides
Py U-and Dab U-modified oligonucleotides (ODNs) were synthesized on a CPG support (1 µmol scale, 1000 Å pore size) using standard phosphoramidite methods and an automated DNA synthesizer (POLYGEN DNA-Synthesizer). The synthesized ODNs were cleaved from the solid support upon treatment with 28-30% aqueous NH 4 OH (1.0 mL) for 12 h at 55 °C. After filtration of the CPG, the crude products from the automated ODN synthesis were lyophilized and diluted with distilled water (1 mL). The ODNs were purified through reverse-phase HPLC (Merck LichoCART C18 column; 10  250 mm; 10 μm; pore size: 100 Å). The HPLC mobile phase was held isocratically for 10 min with 5% MeCN/0.1 M triethylammonium acetate (TEAA) (pH 7.2) at a flow rate of 2.5 mL/min. The gradient was then increased linearly over 10 min from 5 to 50% MeCN/0.1 M TEAA at the same flow rate.
The fractions containing the purified ODNs were cooled and lyophilized. 80% 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 through reversephase HPLC using almost the same eluent system (detection: 254 nm). The products were characterized using MALDI-TOF mass spectrometry.

ODN sample preparation for experiments
For UV spectroscopy, fluorescence spectroscopy, and melting temperature (T m ) and circular dichroism (CD) measurements, 1.0 (UV and fluorescence spectroscopy), 2.0 (T m ), and 3.0 (CD) μM of the ODN was added to a solution of 1 M Tris-HCl buffer (pH 7.2, 100 μL), 1 M NaCl (100 μL), 200 mM MgCl 2 (50 μL) and water (in a 1.5-mL microtube) to give a total volume of 1 mL, followed by vortex-mixing. In the case of duplexes, the probe ODN and the target RNA and/or the quencher ODN were added to 1 M Tris-HCl buffer (pH 7.2, 100 μL), 1 M NaCl (100 μL), 200 mM MgCl 2 (50 μL) and water and then the sample subjected to vortex-mixing. To prepare annealed samples, the mixtures in a buffer solution were heated at 90 °C for 3 min, then slowly cooled under ambient conditions for 4 h.

UV and fluorescence spectra
UV and fluorescence spectra were recorded using Cary 100 and Eclipse spectrometers (Varian), respectively. Samples for UV and fluorescence spectroscopy were prepared in a quartz cell (path length: 1 cm). All samples were measured after baseline correction for UV spectra. Parameters for fluorescence spectra; excitation wavelength: 380 nm, scanning range: 390-700 nm, excitation and emission slits: 5 nm/5 nm; data interval: 1.0 nm.

Melting temperatures (T m )
All values of T m were recorded at 260 nm in a quartz cell (path length: 1 cm) using a Cary 100 Conc UV-Vis spectrophotometer (Varian) equipped with a temperature controller. The values of T m were calculated from the maximum values of the first derivatives in plots of absorbance with respect to temperature.

CD spectroscopy
CD spectra of the ODNs were recorded using a J-810 apparatus (JASCO) equipped with a temperature controller. For each sample, five spectral scans were accumulated at 20 °C over the wavelength range from 220 to 340 nm.

20% Native polyacrylamide gel electrophoresis (PAGE)
5 mL of 40% acrylamide, 2 mL of 5X TBE buffer, and 3 mL of distilled water were mixed for 20% non-denaturing gel. 12 mg of ammonium persulfate was added to the mixed solution.