Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

Abstract Nucleolytic ribozymes utilize general acid‐base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2′‐OH group to attack the scissile phosphate (γ‐catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, “general base knockout” by replacement of the guanine N1 atom by carbon results in only 2.7‐fold decreased rate. Therefore, the common view that γ‐catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ‐catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.


2'-(3''-Azidopropoyl)uridine (3)
Prior to the reaction compound 2 (568 mg, 2.51 mmol) was coevaporated three times with dry pyridine and was stored over phosphorous pentoxide (P2O5) in a desiccator overnight.It was suspended in (4ml) N,N-dimethylacetamide and azidopropanol (1.35 ml, 14.58 mmol, 5.8 eq) as well as boron trifluoride diethyl etherate (1.27 ml, 10.05 mmol, 4 eq) were added.The reaction mixture was heated to 120 °C for 16 hours.All solvents were removed in vacuo and the remaining residue was absorbed on silica in methanol.Purification of the crude product was performed by silica gel column chromatography (methanol/dichloromethane, 0:100-3:97).Compound 3 (148 mg, 452 µmol) was coevaporated three times with anhydrous pyridine and dried on high vacuum overnight.The solid was dissolved in 4.5 ml pyridine and 4,4'-Dimethoxytrityl chloride (168 mg, 497µmol) was added portion wise within 1.5 hours.The solution was stirred at room temperature overnight until the reaction was complete, monitored by TLC.The reaction was quenched with methanol and reduced in vacuo.The residue was diluted with dichloromethane and extracted with 5% citric acid, water and saturated NaHCO3.The crude product was purified by silica gel column chromatography (methanol/ dichloromethane, 0:100-2:98).Compound 4 (162 mg, 258 µmol) was coevaporated three times with anhydrous pyridine and dried over P2O5 overnight.A mixture of anhydrous DMF/pyridine (1:1, 7.4 ml) was added, followed by the addition of 4-(dimethylamino)pyridine (37 mg, 309 µmol) and adipinic acid pentafluorophenyl ester 8443 mg, 927 µmol).The reaction mixture was stirred at room temperature for 1 hour.Then, the solvents were evaporated and the remaining liquid was coevaporated with acetone and dichloromethane.The The crude product was purified by silica gel column chromatography (acetone/dichloromethane, 1:99-5:95).Amino-functionalized solid support (GE Healthcare, Custom Primer Support 200 Amino, 436 mg) was transferred into a syringe equipped with a polypropylene filter.The resin was washed with dry dichloromethane, followed by dry N,N-dimethylformamide.Then, the solid support was suspended and swelled in 2.5 ml N,N-dimethylformamide for 30 minutes.Compound 5 (103 mg, 112 µmol) was dissolved in a small amount of N,N-dimethylformamide.Subsequently the mixture was combined with the resin suspension in the syringe.The suspension was shaken for 48 hours at room temperature.Then, the solvent was filtered off by the syringe and the remaining solid support was washed four times with N,Ndimethylformamide, methanol and dichloromethane, and subsequently allowed to dry.In a final capping step, the resin was treated with a mixture of 3.0 mL Cap A ((acetic anhydride/2,4,6trimethylpyridine/acetonitrile, 2/3/5) and 3.0 mL Cap B (4-(N,N-dimethylamino) pyridine/ acetonitrile, 0.5 M) and was shaken for 4 min at room temperature.Finally, the solid support was washed several times with acetonitrile, methanol and dichloromethane.The product was removed from the syringe and dried under vacuum.

Mass spectrometry
All experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrumentation connected to a Thermo Fisher Ultimate 3000 HPLC system.RNAs were analyzed in the negative-ion mode with a potential of −4 kV applied to the spray needle.LC: Sample (200 pmol RNA dissolved in 30 µL of 20 mM ethylenediamine tetraacetic acid (EDTA) solution; average injection volume: 30 µL); column (Waters XTerraMS, C18, 2.5 µm; 1.0 × 50 mm) at 21°C; flow rate: 0.1 mL/min; eluant A: 8.6 mM triethylamine (TEA), 100 mM 1,1,1,3,3,3hexafluoroisopropanol in H2O (pH 8.0); eluant B: methanol; gradient: 0-100% B in A within 30 min; UV detection at 254 nm. 1  A solution of Sulfo-Cy5-NHS ester (26 mM, 50 µl) was prepared in anhydrous DMSO and of Sulfo-Cy3-DBCO (8.4 mM, 125 µl) in DMSO/water (50% vol/vol), respectively.3'-end 2'-O-(3azidopropyl) RNA (50 nmol) containing a 5'-(6-aminohexyl)-phosphate modification was lyophilized and dissolved in water (30µl).Then, the RNA was desalted by precipitation with sodium acetate buffer (1M, pH 5.3, 0.2 volumes of aqueous solution) and absolute ethanol (3 volumes of aqueous solution) for 2 h, at -20 °C, followed by centrifugation for 30 min at 4 °C at 12,500 RPM (Eppendorf 5430R, rotor F-45-30-11).The pellet was washed with a small amount of ethanol, centrifuged and briefly dried on high vacuum.For the labeling reaction the RNA was dissolved in labeling buffer (sodium borate buffer, 0.1 M, pH 8.5) and DMSO (50% vol/vol) to give a final concentration of 222 µM RNA and 1.6 mM Sulfo-Cy5-NHS ester (26 mM, 13.6 µl) in a total volume of 225 µl.The reaction mixture was shaken overnight in the dark.Subsequently, the RNA was precipitated by the addition of sodium acetate buffer (1 M, pH 5.3, 0.2 volumes of labeling reaction) and ethanol (2.5 volumes of labeling reaction), for 2h at -20 °C.The excess of unreacted hydrolyzed dye was removed by centrifugation for 30 min at 4 °C at 12,500 RPM.For the second labeling step, the single labeled RNA was dissolved in DMSO/water (50% vol/vol) to give a total concentration of 333 µm RNA and 667 µM Sulfo-Cy-3 DBCO (8.4 mM, 14.9 µl) in a final volume of 150 µl.The reaction mixture was shaken for 3 h at room temperature in the dark.Monitoring and purification of the reactions was performed by anion exchange HPLC.In case of incomplete reactions, the reaction mixture was precipitated and the corresponding labeling reaction was repeated without previous HPLC purification steps.

Kinetics of ribozyme cleavage (HPLC assay) 2,3
Nanomole aliquots (2.64 nmol) of the ribozyme and substrate strand were taken from aqueous stock solutions, mixed and lyophilized.The RNA was dissolved in 33.6 µl (78.6 µM) nanopure water, heated to 90 °C for 2 min and allowed to cool to room temperature.For time point 0 (prior to the Mg 2+ induced cleavage reaction) 2.8 µl (220 pmol) of the RNA solution were diluted with nanopure water to a total volume of 100 µl.Further, 6.6 µl HEPES buffer (200mM, pH 7.5) and 2.2 µl KCl solution (2M) were added to the RNA solution.Subsequently, the cleavage reaction was initiated by the addition of 4.4 µl MgCl2 solution (20 mM), leading to total concentrations of 55.0 µM RNA, 30 mM HEPES, 100mM KCl and 2 mM Mg 2+ in a total volume of 44.0 µl.Samples (4 µL) were drawn after the indicated time points, quenched with EDTA solution (4 µl, 40 mM) and diluted with water to a final volume of 100 µl.

Kinetics of ribozyme cleavage (FRET assay)
All measurements were performed on a Cary Eclipse spectrometer (Varian, Australia) equipped with a peltier block and a magnetic stirring device.Equivalent amounts (60 pmol) of Cy5/Cy3 labeled substrate strand and ribozyme strand were lyophilized.The RNA was dissolved in 120 µl MOPS buffer (50 mM KMOPS pH 7.5, 100 mM KCl) to reach a final concentration of 0.5 µM, heated to 90 °C and allowed to cool to room temperature for 10 minutes.The solution was transferred into a quartz cuvette.Subsequently, the fluorescence trace was recorded by using following parameters: excitation: 548 nm, emission: 662 nm data point collection: 0.2 s, slit width 10 nm, detector voltage: 690, temperature: 20 °C.After 1 min of base line detection, the cleavage reaction was manually initiated by the addition of MgCl2 solution (1.2 µl, 1M) to gain a final concentration of 10 mM Mg 2+ .The decreasing FRET intensity was monitored until a constant fluorescence value was achieved as a result of total cleavage of the substrate strand.Three independent measurements were performed for all ribozyme variants of interest.The fluorescence data were fitted by a three-parameter (A1, A2 and kobs) single-exponential equation (1) with A1 as final fluorescence, and A2•exp(-kobs•t) as change in fluorescence over time (t) at the observed rate kobs.Data processing was performed by using the software package OriginPro 2018 (OriginLab, USA).
Determination of pH-rate profiles for ribozyme cleavage (FRET assay/HPLC assay) 4,5,6 The experimental setup was performed as described above (Kinetics of ribozyme cleavage (FRET assay and HPLC assay, respectively)).The pH series for the ribozyme variants were measured in corresponding buffer systems (Supporting Table S1), with three independent measurements for each pH value.The according pH values were fitted against the observed rate constants (kobs) by using a three-parametric (kmax, pKa A , pKa B ) equation ( 2), representing a bell-shaped pH profile, revealing a maximum at (pKa A + pKa B )/2. Kmax reflects the maximum of the cleavage activity in the case of complete protonation of the general acid and complete deprotonation of the general base, independent of the pH value.Consequently, pKa A denotes the pKa of the acidic group that is deprotonated and pKa B denotes the pKa of the basic unit that is protonated, respectively.In the presence of an influencer a cubic cooperative model, reflected by equation ( 3) was applied, including a pKa of the influencer species termed as pKa I , leading to pKa shifts denoted as DpKa coop and two plateaus for the cleavage activity that refer to k1 and k2.As a consequence of the small difference among the apparent pKa values (DpKa~1) the rate-pH profiles remain sharp, limiting the accuracy of the predicted values and errors, respectively.Therefore, we fixed kmax and k1, respectively, to a certain value, that showed the most precise fit for our data points with reliable errors.Data processing was performed by using the software package OriginPro 2018 (OriginLab, USA).