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RNA‐Cleaving Deoxyribozymes Differentiate Methylated Cytidine Isomers in RNA

Abstract Deoxyribozymes are emerging as modification‐specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA‐cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3‐methylcytidine (m3C), N 4‐methylcytidine (m4C), and 5‐methylcytidine (m5C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10‐ to 30‐fold accelerated cleavage of their target m3C‐, m4C‐ or m5C‐modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC‐detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3C and m5C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites.


Synthesis of m 3 C phosphoramidite
All synthetic procedures were performed under nitrogen atmosphere. The chemicals were purchased 'Pro analysis'-or 'For synthesis' grade and used without additional purification. Dry solvents (DCM, MeCN, DMF, THF) were obtained in a solvent purification system (SPS). DCE was dried over molecular sieves. Solvents for column chromatography (technical quality) were distilled prior to use. The obtained compounds were stored at 4 °C (-20 °C in case of TOM-protected compounds and the final phosphoramidite). Column chromatography was performed on silica gel (Kieselgel 60, Merck, 0.040-0.063 mm). TLC was performed on Alugram DIL G/UV254 (Machery-Nagel, UV visualization, 254 nm). NMR spectra were recorded on a Bruker Avance III HD 400 spectrometer. Chemical shifts are reported relative to the residual solvent signals. The synthesis of m 3 C-phosphoramidite building block 5 was performed in five steps. The methyl group was introduced at position N3 of cytosine with dimethyl sulfate, [4] resulting in formation of salt 1. The transient protection of the ribose hydroxy groups with TMSCl, allowed selective protection of the amino group with benzoyl chloride to obtain compound 2. [5] In the next step, the 2'-and 3'-hydroxy groups of compound 2 were transiently protected as acetal with DMF-DMA, and the 5'-hydroxy group was selectively protected upon reaction with DMT-Cl. Compound 3 was then activated with tBu 2 SnCl 2 and treated with TOM-Cl to yield compound 4. Finally, compound 4 was treated with CEP-Cl to afford m 3 C phosphoramidite 5.

Solid-phase synthesis of oligoribonucleotides
RNA oligonucleotide syntheses were carried out at 0.7-1 μmol scale on ABI 394 DNA/RNA synthesizer using standard -cyanoethyl phosphoramidite chemistry with the building blocks mentioned above. Deprotection of m 3 C-modified oligoribonucleotides was performed with a mixture of concentrated aqueous NH 3 and EtOH (3:1) at 55 °C for 6 h followed by incubation with 1 M TBAF in THF under standard conditions. Conversion of the O 4 -chlorophenyluridine-modified oligoribonucleotide into the m 4,4 C-modified RNA (Table S1, R27) was performed by incubation of the solid support with 0.7 ml of 40% aqueous Me 2 NH at 37°C for 5 h followed by addition of 0.5 ml of aqueous NH 3 and further incubation at 55 °C for 2 h to ensure complete deprotection of cyanoethyl and acetyl groups. Deprotection of TOM groups was achieved with TBAF in THF under standard conditions. Deprotection of other oligonucleotides was performed under standard conditions with MeNH 2 in aqueous ethanol and TBAF in THF. The crude oligonucleotides were desalted by size exclusion chromatography on GE Healthcare Life Sciences ÄKTA™ start system (3x5 ml HiTrap columns), purified by PAGE, and analyzed by anion exchange HPLC and HR-ESI-MS.
Labeling of RNA at 3'-end Unfunctionalized oligoribonucleotides were labeled by periodate oxidation followed by a reaction with fluorescein-5-thiosemicarbazide. In a typical procedure, RNA (0.3-1 nmol) was dissolved in 7.5 μl water, followed by addition of 5x sodium phosphate buffer (2 μl, 250 mM, pH 7.4) and a freshly prepared aqueous solution of NaIO 4 (0.5 μl, 400 mM). The resulting mixture was incubated for 10-15 min at 37 °C. The excess of NaIO 4 was quenched by treatment with aqueous Na 2 SO 3 (1 μl, 1 M) for 5-10 min at 37 °C. Afterwards, a solution of fluorescein-5-thiosemicarbazide (1 μl, 10 mM) in DMF was added, and the reaction mixture was incubated in the dark for additional 1 h at 37 °C. The labeled product was then purified by PAGE (10-20% polyacrylamide) and recovered by extraction and ethanol precipitation.

Splinted ligation of RNA substrate and deoxyribozyme library
Splinted ligation was carried out with T4 DNA ligase as described earlier. [7] In a typical procedure, the deoxyribozyme pool, 5'-phosphorylated RNA and complementary DNA splint (1.8 nmol, 2.4 nmol and 2 nmol, respectively) were dissolved in water (9 μl), and 10x annealing buffer (1 μl; 40 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) was added. After 2 min incubation at 95 °C, the solution was allowed to cool down to r.t. for 15 min and then further cooled down to 0 °C for 10 min. 10x DNA Ligase buffer (2 μl; 400 mM Tris-HCl, 100 mM MgCl 2 , 100 mM DTT, 5 mM ATP, pH 7.8), T4 DNA Ligase (2 μl; 5 U/μl) and water to a total volume of 20 μl were added, and the resulting mixture incubated for 2 h at 37 °C to yield a DNA-RNA hybrid product which was purified by PAGE.

Intramolecular cleavage of RNA-DNA hybrids (key selection step)
The DNA-RNA hybrid (250 pmol in first round and 10-30 pmol in further rounds) was incubated with 10x selection buffer (1 µl, 500 mM Tris-HCl, 1.5 M NaCl, pH 7.5) in a total volume of 9 µl. To initiate the reaction, MgCl 2 (1 µl, 100 mM) was added to the final concentration of 10 mM and reaction mixture was incubated at 37 °C for 16 hours. Active fraction of DNA enzymes was separated using denaturing PAGE (10%) and the gel areas corresponding to the cleaved products (determined by comparison with a size marker) were cut and extracted. Extracted products were ethanol precipitated and amplified with PCR.

PCR amplification of the enriched DNA library
The enriched DNA library was amplified via two subsequent asymmetric PCR reactions. [7] In the first PCR, the extracted product from previous step was used as a template. Template (30 µl), forward primer D2 (200 pmol), reverse primer D3 (50 pmol), dNTP mixture (0.625 µl, 20 mM each dNTP), 10x DreamTaq buffer (5 µl) and DreamTaq DNA polymerase (0.25 µl, 5 U/µl) were mixed, and the total volume was adjusted to 50 µl with water. The resulting mixture was subjected to a 10-cycle PCR reaction (95 °C for 4 minutes, 10× [95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min] and 72 °C for 5 minutes) using a Bio-Rad thermal cycler. For the second PCR reaction, an aliquot (1-2 µl) from PCR-I was used as template. This aliquot was mixed with fluorescently labeled forward primer D2.1 (200 pmol), reverse primer D3 (50 pmol), dNTP mixture (0.6 µl, 20 mM), 10x DreamTaq buffer (5 µl), DreamTaq DNA polymerase (0.5 µl, 5 U/µl) and water to a final volume of 50 µl. The resulting solution was subjected to a 30-cycle PCR reaction (95 °C for 4 minutes, 30x [95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min], and 72 °C for 5 min). The PCR product was subjected to denaturing PAGE and the short strand with the fluorescent label was isolated. The resulting ssDNA was used for ligation to RNA substrate to initiate the next round of selection.

Cloning and sequencing
After 18 rounds of selection, the enriched libraries AK, AL, AM and AN were cloned using TOPO-TA cloning and transformed into E.Coli. The plasmids of 40 randomly picked colonies were isolated and sequenced using Sanger sequencing. Enriched libraries from rounds 7 and 18 were also subjected to deep sequencing. For this purpose, total eight selection libraries were amplified in a 2-step PCR protocol, in which Illumina adaptors and unique indices were introduced. The PCR products were purified on PAGE (10%) and with ZYMO Oligo Clean & Concentrator kit. After quality control on a Bioanalyzer, the libraries were pooled, and sequenced as a fraction of a NextSeq 500 High Output 75 nt single-end run in the Core Unit Systems Medicine (University of Würzburg). After demultiplexing, the resulting fastq files were further processed using custom bash, R and Python scripts.

Kinetics characterization of deoxyribozyme-catalyzed RNA cleavage
Kinetics characterization was carried out as described previously [8] to analyze trans-activity of individual deoxyribozymes or active deoxyribozyme pools under single turnover conditions. In a typical procedure, a deoxyribozyme (100 pmol) was mixed with a fluorescently labeled RNA substrate (10 pmol) in a final volume of 8 µl. The resulting mixture was heated to 95 °C for 4 min and then cooled to 25 °C for 10 min. To initiate the reaction, 10x kinetic assay buffer (1 µl, 500 mM Tris-HCl, 1.5 M NaCl), pH 7.5) and MgCl 2 (1 µl, 200 mM) were added and the resulting mixture was incubated at 37 °C. Aliquots (1 µl) were taken at different time points (e.g. 0, 15 min, 30 min, 60 min, 180 min, and 360 min), quenched with loading buffer and analyzed by denaturing PAGE. Cleavage yields were determined by measuring fluorescent intensities of the corresponding bands. Values of k obs (observed cleavage rate) and Y max (maximum fraction cleaved) were obtained by fitting fraction cleaved versus time with the first-order kinetics equation Y = Y max * (1 − e −kobs*t ). Each experiment was repeated at least trice, and values are reported as mean of three individual experiments.

Synthesis and analysis of mt-tRNAs
Unmodified mt-tRNA Glu was prepared by in vitro transcription from a synthetic DNA template using T7 RNA polymerase. Reactions were performed in aqueous solution containing 40 mM Tris-HCl, pH 8.0, 10mM DTT, 4 mM each NTP, 30 mM MgCl 2 and 2 mM spermidine and 20 µg of T7 RNA polymerase (prepared in house) at 37 °C for 5 h. The transcription products were purified by denaturing PAGE (10% polyacrylamide) and dephosphorylated by calf intestinal alkaline phosphatase. The m 5 C-modified mt-tRNA Glu was assembled by splint ligation of two RNA fragments (R25 and R26). The m 3 C-modified mt-tRNA Thr and unmodified mt-tRNA Thr were constructed by splint ligation of three synthetic RNA fragments (R21, R22, R23) and (R21, R22, R24) respectively. In a typical procedure, RNA fragments and complementary splint (D52/D53) in equimolar ratio were annealed in 10x annealing buffer (40 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) and ligation reactions were then performed with T4 DNA ligase for 16 h at 37 °C. Full length products were separated by PAGE (10% polyacrylamide), and labeled with γ-32 P-ATP at their 5'end.Aliquots of the full-length tRNA samples were then subjected to DNAzyme-mediated cleavage using the corresponding DNAzymes (D54/D55/D57/D58) as described above.