Enzymatic incorporation and utilization of an emissive 6-azauridine

Abstract

To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.

Fig. 1

a) Nucleoside design: enhancing the polarization of the conjugated electron poor/electron rich biaryl system. b) Modified uridines: 5-(thiophen-2-yl)-6-azauridine (1), 6-azauridine (2), 5-(thiophen-2-yl)uridine (3) and their corresponding triphosphates 4, 5, 6.

Fig. 2

(a) Transcription reaction with T7 promoter and template A in the presence of natural NTPs and UTP or modified UTPs 4–6; resulting in transcripts 7–10 respectively. Transcription reaction with T7 promoter and template B in the presence of natural NTPs and UTP or modified 4, resulting in transcripts 11, 12 respectively. (b) Ligation reaction with T4 DNA ligase, modified acceptor, donor and splint.

Fig. 3

Transcription reactions were carried out with T7 promoter and template A in the presence of natural NTPs and UTP or modified UTPs 4, 5, 6. Lane 1: control reaction with all natural NTPs; lane 2: competition reaction in the presence of equimolar concentration of UTP and 4, lane 3: reaction in the presence of 4, lane 4: reaction in presence of 4 (*5mM instead of 1mM, as for all other reactions), lane 5: reaction in the presence of equimolar concentration of UTP and 5, lane 6: reaction in the presence of 5, lane 7**: reaction in the presence of equimolar concentration of 4 and 5, lane 8: reaction in the presence of 6. The reaction was resolved by gel electrophoresis on a denaturing 20% polyacrylamide gel; under UV light at a) 254 nm (on TLC plate); b) 365 nm; c) black and white picture after Stains-All was used to visualize the oligonucleotides.

Fig. 4

HPLC profile of enzymatic digestion reactions. (a) A mixture of nucleosides used as a standard and (b) digestion results of ligation product 14. Digestion of 1−2 nmol of transcript was carried out using S1 nuclease for 2 h at 37 °C and followed by dephosphorylation with alkaline phosphatase for 2 h at 37 °C. The ribonucleoside mixture obtained was analyzed by reverse-phase analytical HPLC with Agilent column eclipse XDB-C18 (5μm, 4.6 × 150mm). Mobile phase: 0−5% acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 10 min and then increase 5−35% acetonitrile (0.1% formic acid) in water (0.1% formic acid) 10−17 min; flow rate 1 mL/min.

Fig. 5

(a) Binding of aminoglycosides to a fluorescently modified A-site hairpin 14. (b) Binding and displacement events of A-site bound coumarin-aminoglycoside with unlabelled RNA binders (where 6-aza modified uridine 1 is a FRET donor and coumarin is a FRET acceptor).

Fig. 6

Fluorescence titrations of the A-site construct 14 with neomycin (15). (a) Representative emission spectra for titrations of A-site hairpin 14 with neomycin. (b) Curve fit for the titrations of A-site hairpin 14 with neomycin (15) with respective values EC₅₀ 0.9 ±0.2 × 10⁻³ mM and 2 ±1 × 10⁻² mM (fractional saturation vs. antibiotic concentration). (c) Representative emission spectra for displacement studies: titrations of A-site hairpin 14 with neomycin (15). (d) Curve fit for the displacement studies: titrations of A-site hairpin 14 with neomycin with respective values EC₅₀ 3.0 ±0.1 × 10⁻³ mM and 3 ±1 × 10⁻² mM [fractional saturation vs antibiotic concentration; 1, FRET donor (purple), 6″-amino-6″-deoxykanamycin-coumarin conjugate, FRET acceptor (green)]. Each event was fitted separately (see SI for details).

Fig. 7

Normalized absorption (---) and emission (—) spectra of 5-thiophene-6-azauridine in purple (1; FRET donor) and coumarin in green (FRET acceptor).

Scheme 1

Synthesis of modified nucleoside triphosphate 4. Reagents and conditions: i) POCl₃, (MeO)₃PO; ii) tributylammonium pyrophosphate, proton sponge, 0–4°C. See Supporting Information for details.