Amine‐to‐Azide Conversion on Native RNA via Metal‐Free Diazotransfer Opens New Avenues for RNA Manipulations

Abstract A major challenge in the field of RNA chemistry is the identification of selective and quantitative conversion reactions on RNA that can be used for tagging and any other RNA tool development. Here, we introduce metal‐free diazotransfer on native RNA containing an aliphatic primary amino group using the diazotizing reagent fluorosulfuryl azide (FSO2N3). The reaction provides the corresponding azide‐modified RNA in nearly quantitatively yields without affecting the nucleobase amino groups. The obtained azido‐RNA can then be further processed utilizing well‐established bioortho‐gonal reactions, such as azide‐alkyne cycloadditions (Click) or Staudinger ligations. We exemplify the robustness of this approach for the synthesis of peptidyl‐tRNA mimics and for the pull‐down of 3‐(3‐amino‐3‐carboxypropyl)uridine (acp3U)‐ and lysidine (k2C)‐containing tRNAs of an Escherichia coli tRNA pool isolated from cellular extracts. Our approach therefore adds a new dimension to the targeted chemical manipulation of diverse RNA species.

To remove cyanoethyl groups and the N-Fmoc group of the assembled aminoacyl-3'-NH-RNAs, the support (~25 mg) was kept in the synthesis cartridge and rinsed with 20 mL of 20% piperidine in acetonitrile followed by 20 mL of acetonitrile.Then, for base deprotection and cleavage from the support, the beads were transferred into an Eppendorf tube and equal volumes of CH3NH2 in ethanol (8 M, 650 µL) and CH3NH2 in H2O (40%, 650 µL) were added.The mixture was kept at room temperature for 8 hours.After that, the supernatant was filtered and evaporated to dryness in a SpeedVac concentrator.
For base deprotection and cleavage from the support of 5-aminomethyl uridine containing RNAs, the support-bound protected RNAs were treated with a mixture of 40% aqueous methylamine and 30% aqueous ammonia (600 µL, 1:1 v/v) in a screw-cap vial at 65 °C for 15 minutes.Then, the supernatant was filtered and reaction mixture were evaporated to dryness in the SpeedVac.
The 2'-O-silyl (TOM or tBDMS) protecting groups were removed by incubation of oligoribonucleotides in a mixture of N-methyl-2-pyrrolidone (100 µL) and 1 M TBAF trihydrate in THF (1000 µL) at 37 °C for 16 h.The reaction was quenched by addition of 1 M triethylammonium acetate, pH 7.4 (1000 µL) and then concentrated to approximately 0.5 to 1.0 mL.The resulting mixture was desalted on a C18 Sep-Pak® cartridge according to a protocol recommended by the manufacturer (Waters Corporation).The crude oligonucleotide products were evaporated and stored at -20 °C.
The desired oligoribonucleotides were purified and isolated by semi-preparative anion-exchange HPLC on a Dionex DNAPac PA-100 column (9 x 250 mm).Conditions: flow rate 2 mL min -1 ; for eluents and UV detection see above; the gradient was optimized according to the length of the RNA and typically D15% B in 30 min.
The fractions corresponding to a product were desalted using the C18 Sep-Pak® cartridges (Waters Corporation).The quality of the product was analyzed by anion-exchange HPLC and reversed-phase LC-ESI MS.Sequences and mass spectrometric data for all the obtained RNAs are shown in Supporting Table S1.

High-resolution mass spectrometry of compounds
High resolution mass spectra were recorded in positive ion mode on a Thermo Scientific Q Exactive Orbitrap, ionized via electrospray at 3.7 kV spray voltage.

Preparation of fluorosulfuryl azide (FSO2N3)
The diazotizing reagent, fluorosulfuryl azide (FSO2N3), was essentially prepared according to the procedure described in [2] and optimized as described in the following.To a stirred biphasic system of aqueous NaN3 solution (97.5 mg, 1.50 mmol, 3 ml) and MTBE (3 mL) in a loosely sealed plastic bottle, cooled in an ice-water bath, was rapidly added a solution of 1-(fluorosulfuryl)-2,3-dimethyl-1Himidazol-3-ium trifluoromethanesulfonate (0.5 g, 1.52 mmol; CAS 2179072-33-2)) in acetonitrile (0.25 mL).The reaction was stirred vigorously for 10 min at 0 °C and then rested at room temperature for 30 min.The organic phase containing FSO2N3 was separated from the aqueous phase, and it was kept in a loosely sealed plastic bottle at room temperature.After the 12-hour resting period, the organic phase was transferred to Eppendorf ultracentrifuge 2 ml tubes and centrifuged at 13,400 rpm for 15 min, to facilitate separation of the phases.The colorless solution of FSO2N3 in MTBE was separated from the pink residual aqueous phase and was used for the diazotransfer reaction directly without further purifications.The concentration and yield of the FSO2N3 solution were measured by 19 F NMR as described in [2], giving the values of 450 to 480 mM and ~90%, respectively.

Diazotransfer reaction on RNA using FSO2N3
For the diazotransfer reaction, an equivalent of RNA containing a primary amino group (final concentration in a range of 20-50 µM) was dissolved in 0.1 M NaHCO3, pH 8.3 (100 µL) and DMF (20 µL), after that two equivalents of FSO2N3 in MTBE (90 to 210 µL) were added, and the mixture was thoroughly mixed (1.400 rpm) for 20 min at room temperature.Then, to facilitate separation of the phases, the reaction mixture was centrifuged at 13,400 rpm for 10 min.The colorless organic phase was removed from residual aqueous phase containing the RNA products.The azide-modified oligoribonucleotides were precipitated with 2% NaClO4 in acetone (1000 µL), the resulting pellet was washed with acetone (600 µL) and air-dried.According to their AE-HPLC profiles, the conversion for all the reactions was >90% (see Supporting Table 1, Figure 2 and Supporting Figure 3).

Labeling of RNAs with biotin-PEG4-alkyne by copper(I)-catalyzed cycloaddition (CuAAC)
For the CuAAC, the copper(I)-catalyst was generated in situ from a complex of CuSO4 and watersoluble tris(3-hydroxypropyltriazolylmethyl)amine (THPTA ligand) [3] using freshly prepared solution of sodium ascorbate as a reducing agent.In brief, a 70 mM solution of THPTA ligand (50 µL) was mixed with a 0.1 M solution of CuSO4•5H2O (5 µL), purged with a slow flow of argon for 1 min, and mixed with sodium ascorbate (50 µL; 1 M aqueous solution).This solution was added to the degassed mixture of annealed azido modified oligoribonucleotide (2-6 nmol in 20 µL of deionized H2O) and biotin-PEG4-alkyne (0.4 mg, ~1 µmol) in DMF (20 µL).The reaction mixture was degassed again and kept at room temperature for 1 h.After that, the oligoribonucleotides were precipitated with 2% NaClO4 in acetone (1000 µL), the resulting pellet was washed with acetone (600 µL) and air-dried.The oligoribonucleotide material was dissolved in water (400 µL), and Vivaspin 500 Centrifugal Concentrators (Sartorius) were used to remove reagents from oligonucleotide material.According to the AE-HPLC profiles, a conversion for all of the reactions was in a range of 78-98 % (see Figure 3A and Supporting Figure 4, Supporting Table 1).

RNA-peptide conjugates by traceless Staudinger ligation
For the traceless Staudinger ligation, azido-modified oligonucleotide was converted to a form soluble in organic solvents.For this purpose, an oligoribonucleotide (2-6 nmol in 50-100 µL of deionized H2O) was precipitated by 8% cetyltrimethylammonium bromide (CTAB), with a few portions of 2 µL each.After addition of each CTAB portion, the mixture was vortexed and centrifuged (2 min, 13400 rpm).The precipitation by CTAB was continued until the solution ceased to cloud with the addition of CTAB reagent.Then, the remaining water was removed, and the pellet was dissolved in DMF (100 µL) at 60 °C.The PPh3OfMet ester (4.4 mg, 10 µmol) was added to the resulting solution of the cetyltrimethylammonium salt of the azido modified oligoribonucleotide in DMF.The reaction mixture was stirred for 4 h at 60 °C.After that, the oligoribonucleotide was precipitated with 2% NaClO4 in acetone (1000 µL), the resulting pellet was washed with acetone (600 µL) and air-dried.According to the AE-HPLC profiles, a conversion for the reaction varied from ~60-86 % depending on the RNA conjugate (see Figure 3B and Supporting Figure 4, Supporting Table 1).

Pull-down of E. coli tRNAs containing nucleotide modifications with primary amino groups
Labeling.Total tRNA from E. coli (~1 mg, Sigma-Aldrich) was dissolved in deionized H2O (88 µL) and DMF (20 µL), incubated at 80 °C for 3 min, and then immediately chilled on ice.A solution of 1 M NaHCO3 (12 µL, to final concentration of 0.1 M, pH 8.3) was added to the mixture.Thereafter, FSO2N3 in MTBE (200 µL) was added, and the mixture was thoroughly mixed (1.400 rpm) for 20 min at room temperature.The reaction mixture was centrifuged at 13,400 rpm for 10 min.The colorless organic phase was removed and the tRNA pool was precipitated from the residual aqueous phase by adding 3 M sodium acetate (10 µL, pH 5.2) and ice-cold absolute ethanol (600 µL), the resulting pellet was washed with ice cold 70 % ethanol (400 µL) and air-dried.The following labeling of the tRNA pool with desthiobiotin-PEG4-alkyne (Jena Bioscience) was performed according to the procedure described above in Section 5.After that, the Vivaspin 500 Centrifugal Concentrators were used to remove reagents from the tRNA material.
For analysis of the complex reaction mixture after the diazotransfer and CuAAC reactions, the E.coli tRNAs were directly labeled with commercial alkyne-modified 5-carboxytetramethylrhodamine dye (F545) and subjected to denaturing polyacrylamide gel electrophoresis (in 10 % PAAG (19:1), 8 M urea).A fluorescent band of the F545-labeled tRNAs was observed with the expected electrophoretic mobility similar to the unmodified tRNA reference, consistent with successful labeling of the azidomodified tRNAs by the CuAAC reaction without RNA degradation (Supporting Figure S7).
Immobilization.The Streptavidin Magnetic Particles (magnetic beads, Sigma-Aldrich) were thoroughly mixed and a 200 µL aliquot of the beads was taken for capturing of the biotinylated tRNA species.The supernatant was removed and the beads were washed twice with a solution of 100 mM NaOH and 50 mM NaCl (300 µL), then washed once with a solution of 100 mM NaCl (300 µL), followed by washing twice with a binding buffer consisting of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl (200 µL).After that, the binding buffer (200 µL) was added and the beads were resuspended.The labeled tRNA pool was added to the suspension of the beads, the mixture was gently mixed by pipetting, and incubated at 25 °C for 30 min.The supernatant was removed and the beads were washed twice with 200 µL of 20 mM Tris-HCl (pH 7.5).

Non-denaturing elution.
A solution of biotin (Sigma-Aldrich) (~5 mg/mL) was added to the tRNA modified beads.The suspension was mixed by pipetting and incubated at room temperature for 30 min with gentle shaking to prevent sedimentation of the beads.Next, the beads were centrifuged at 2000×g for 30 s, and the supernatant was collected.The elution was repeated three more times.After that, the Vivaspin 500 Centrifugal Concentrators were used to remove reagents from the collected supernatant containing tRNA material.The tRNA pool was precipitated from the residual aqueous phase by adding a solution of 3 M sodium acetate (10 µL, pH 5.2) and ice cold absolute ethanol (600 µL), the resulting pellet was washed with ice cold 70% ethanol (400 µL) and air-dried.
For schematic representation of the tRNA enrichment procedure and the cloverleaf structures of all the E. coli tRNAs containing acp 3 U47 see Figure 4 and Supporting Figure S6, respectively.

Northern blotting assay to identify tRNA targets having primary amino groups
The tRNA pool obtained after the tRNA enrichment procedure (~20 µg) was heated at 80 °C in a mixture containing 8 M urea, 0.05% xylene cyanol FF and 0.05% bromophenol blue for 2 min, then cooled to room temperature, and loaded on a denaturing 10% polyacrylamide gel (19:1, 8 M urea; ~2 µg/pocket).
The transcribed tRNA Lys was used in the experiment as an RNA ladder.Following standard capillary transfer procedures, tRNA pool was transferred in 0.5x TBE buffer (0.05 M Tris base, 0.05 H3BO3, and 1 mM EDTA, pH 8.3) to a positively charged nylon membrane (Hybond ® -N+ hybridization membrane).Subsequently, tRNAs were immobilized on the membrane by irradiation in the BioDocAnalyze (Biometra) for 2 min.After 60 min of prehybridization in a hybridization buffer (1 M sodium phosphate buffer pH 6.2, 7 % SDS), a 5′-32 P-labeled probe (prepared with [gamma -32 P]ATP and T4 polynucleotide kinase according to standard procedures) complementary to a variable region of the respective tRNAs from E. coli (20 pmol, see sequences in Supporting Table 2) was added and the blot was incubated overnight at 42 °C.Next, the blot was washed in a washing buffer I (0.1 % SDS, 0.6 M NaCl, 0.06 M trisodium citrate, pH 7.0) for 3 min at 25 °C, then in a washing buffer II (0.1 % SDS, 0.03 M NaCl, 3 mM trisodium citrate, pH 7.0) for 2 min and rinsed in water.The membrane was air-dried and placed in an X-ray cassette, where it was exposed for 24 h.Visualization of bands was performed using Typhoon FLA 9500 phosphorimager (GE Healthcare).Supporting Figure S1.Diversity of known natural RNA modifications having a primary aliphatic amino group and found in rRNAs and tRNAs [4].Supporting Figure S3.Anion-exchange HPLC and mass spectrometric analysis of a reference RNA (5'-AACGAGGCCACAGG-3') having no aliphatic primary amino groups after being exposed to the optimized diazotransfer reaction conditions.The molecular weight is calculated for the intact reference RNA.The RNA is stable under these conditions and no reaction occurs at the nucleobase amino groups.S1

.
Overview of amino-and azido-modified oligoribonucleotides and conjugates used in this study.Oligonucleotide sequence in 5' to 3' direction and peptide sequence from C to N terminus; b determined from areas in HPLC profiles; c AE-HPLC retention time (RT); d molecular weights m.w.obtained by LC-ESI ion trap mass spectrometry. a

Table S2 .
Synthetic DNA and 2'-OCH3 oligonucleotides used as probes for identification of specific E. coli tRNAs by Northern blotting.