RNA Probes for Visualization of Sarcin/ricin Loop Depurination without Background Fluorescence

Abstract Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase (depurination) at the sarcin/ricin loop of the ribosomal RNA. In this work, we describe the preparation and optimization process of a fluorescent probe that can be used to visualize depurination. Starting from a fluorescent thiophene nucleobase analog, various RNA probes that fluoresce exclusively in the presence of a depurinated sarcin/ricin‐loop RNA were designed and characterized. The main challenge in this process was to obtain a high fluorescence signal in the hybridized state with an abasic RNA strand, while keeping the background fluorescence low. With our new RNA probes, the fluorescence intensity and lifetime can be used for efficient monitoring of depurinated RNA.

The solvent was removed under reduced pressure and the crude product was purified via column chromatography (DCM/MeOH 95:5, 1% NEt3). Nucleoside 4 was obtained as a white foam. Compound 5: Synthesis was performed according to literature. [2] Nucleoside 4 (237 mg, 0.39 mmol, 1 eq) was dissolved in 5 mL anhydrous dichloroethane. Compound 6: The synthesis was performed referring to a procedure known from literature. [2] TOMprotected nucleoside 5 (160 mg, 0.20 mmol, 1 eq) was dissolved in 5 mL dry dichloromethane. Diisopropylethylamine (96 mg, 0.41 mmol, 2 eq) and 2cyanoethoxy-N,N-diisopropylaminochlorophosphine (131 mg, 1.01 mmol, 5 eq) were added subsequently under continuous stirring. The mixture was stirred at room temperature for 20 hours under an argon atmosphere. The solvent was removed under reduced pressure and the crude product was purified via column chromatography (cyclohexane/EtOAc, 2:1). The product was obtained as a white foam.

Solid-phase synthesis:
Solid-phase synthesis was performed on an ABI392 instrument.  3 and MeOH (Fluka) were used with a gradient from 5% to 100% MeOH in 22 minutes. The 5' DMTr-protecting groups were removed by incubating the oligonucleotides with 80% AcOH (1 ml) at room temperature for 20 minutes. The solvents were removed using a vacuum concentrator and the DMTr-off oligonucleotides were purified via reverse phase HPLC using the same conditions as described above.
Quencher labeling: The oligonucleotide was dissolved in borate buffer (pH 8.45). A 60-fold excess of the Dabcyl-NHS (Sigma-Aldrich) in DMSO was added. The volume ratio of buffer to DMSO was 3:1. After incubation for approximately 16 hours at 35 °C, a 60-fold excess of the quencher in DMSO was added again and buffer was supplemented so that the 3:1 ratio was maintained. After incubating again for eight hours at 35 °C, additional quencher in DMSO and buffer was added a third time. After final incubation for another 16 hours at 35 °C the excess of quencher was removed by size exclusion chromatography via Sephadex™ G-25 M from GE Healthcare. The oligonucleotides were purified via reversed phase HPLC with a gradient from 5% to 50% MeOH in 13 minutes. Table S1. Sequences and determined molecular masses of the synthesized oligonucleotide probes 1-8 and hybridization strands SRL and SRL abasic.

Fluorescence measurements
For steady state fluorescence intensity measurements the final concentration of the probes was 10 µM (n = 1 nmol) and for the counterstrands SRL and SRL abasic 20 µM (n = 2 nmol). The final volume used was 100 µl and the solutions were prepared in CSH Brain Buffer, having a final salt concentration of 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, and 5 mM HEPES at pH 7.4. Measurements were performed on a Tecan infinite M200PRO plate reader at 37 °C. 304 nm was used for excitation and fluorescence intensity at 408 nm was used for evaluation. Every measurement was repeated 3-5 times.
The time-correlated single photon counting (TCSPC) experiments were conducted with an FT100 spectrometer (PicoQuant, Berlin). For excitation, a pulsed LED PLS310 with a central wavelength of 310 nm and a pulse duration of 800 ps was applied, controlled by a PDL800-D driver (PicoQuant, Berlin). Time-resolved fluorescence measurements were acquired with software TimeHarp260 (PicoQuant, Berlin). The instrument response function (IRF) was measured by the scattered light of TiO2 dispersed in ethanol. For the sample fluorescence measurements, a UVB390 filter was used to cut off the excitation stray light and the excitation. The concentrations of the labeled probes were 2 µM and the counter strands were provided in excess (3 µM). All of the samples were prepared in CSH brain buffer in 4x10 mm quartz glass cuvettes. Exponential fitting of the data was performed with the software FluoFit 4.6 (PicoQuant, Berlin). [3] Figure S1. Fluorescence decay curves of the free probes (magenta), the hybridized states with SRL (gray) and with SRL abasic (green) of the probes 1-8. The semi-transparent dots represent the data points and the solid line the obtained multiexponential fits. Fluorescence measurements with full-length SRL and full-length SRL abasic: Figure S2. Probes 3, 4, 5 and 7 and their fluorescence properties when hybridized to complementary full-length SRL and full-length SRL abasic RNA. cprobe = 10 µM in CSH brain buffer (135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 5 mM HEPES) at 37 °C, 2 eq. counter strand RNA, λex = 304 nm, vges = 100 µL.