Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19 F NMR Spectroscopy

Abstract

Development of versatile probes that can enable the study of different conformations and recognition properties of therapeutic nucleic acid motifs by complementing biophysical techniques can greatly aid nucleic acid analysis and therapy. Here, we report the design, synthesis and incorporation of an environment-sensitive ribonucleoside analogue, which serves as a two-channel biophysical platform to investigate RNA structure and recognition by fluorescence and ¹⁹ F NMR spectroscopy techniques. The nucleoside analogue is based on a 5-fluorobenzofuran-uracil core and its fluorescence and ¹⁹ F NMR chemical shifts are highly sensitive to changes in solvent polarity and viscosity. Notably, the modified ribonucleotide and phosphoramidite substrates can be efficiently incorporated into RNA oligonucleotides (ONs) by in vitro transcription and standard solid-phase ON synthesis protocol, respectively. Fluorescence and ¹⁹ F readouts of the nucleoside incorporated into model RNA ONs are sensitive to the neighbouring base environment. The responsiveness of the probe was aptly utilized in detecting and quantifying the metal ion-induced conformational change in an internal ribosome entry site RNA motif of hepatitis C virus, which is an important therapeutic target. Taken together, our probe is a good addition to the nucleic acid analysis toolbox with the advantage that it can be used to study nucleic acid conformation and recognition simultaneously by two biophysical techniques.

Keywords: NMR; fluorescence; nucleoside probe; ribonucleoside analogue; viral RNA.

Figure 1

(A) UV (25 μM, solid lines) and steady-state fluorescence (5 μM, dashed line) spectra of nucleoside 1 in solvents with different polarity. In fluorescence study, samples were excited at respective lowest energy absorption maximum with excitation and emission slit width 2 nm and 3 nm, respectively. (B) Steady-state fluorescence spectra (5 μM, dashed line) of nucleoside 1 in solvents with similar polarity (ethylene glycol and glycerol) but different viscosity. Samples were excited at respective lowest energy absorption maximum with excitation and emission slit widths of 2 nm and 2 nm, respectively.

Figure 2

¹⁹F NMR spectra of nucleoside 1 (600 μM) in solvents of different polarity and viscosity. Each NMR sample contained 15% DMSO-d6 and each spectrum was referenced relative to an external standard trifluorotoluene (TFT: -63.72 ppm).

Figure 3

Incorporation of the modified nucleotide analog into RNA ONs by transcription reaction in presence of different DNA templates T1–T7. RNA ON 11 was synthesized by solid phase method using phosphoramidite 12 (Scheme 2). ONs 4c, 9c, 10c and 11c are cDNA ONs used in fluorescence and ¹⁹F NMR studies. "r" represent the RNA ONs and "d" represents DNA ONs.

Figure 4

(A) Polyacrylamide gel electrophoresis of transcripts obtained from in vitro transcription reactions with DNA templates T1–T5 in the presence of UTP and or 2 under denaturing conditions. All reactions were performed in duplicate and standard deviations in yields are ≤ 4%. (B) RP-HPLC chromatogram of a mixture of standard nucleosides (rC, rU, rG, rA) and modified nucleoside 1 (top) and ribonucleoside products obtained from digestion of transcript 4 at 260 nm (bottom). See experimental section for details.

Figure 5

(A) Representative UV-thermal melting profile of control (3•4c) (5 μM) and modified (4•4c) (5 μM) duplexes in sodium phosphate buffer (pH 7.1) containing 500 mM NaCl. (B) Emission spectra (0.5 μM) single stranded ONs 4, 9–11 (solid lines) and duplexes (dotted lines). (C) Bar diagram showing the fluorescence intensity of ONs at respective emission maximum. (D) ¹⁹F NMR spectra of ONs 4, 9–11 and duplexes (50 μM) in sodium phosphate buffer containing 500 mM NaCl.

Figure 6

(A) Crystal structure (PDB ID: 2NOK) of L-shaped bent conformation of IRES subdomain IIa stabilized by Mg^II and Mn^II. (B) Crystal structure (PDB ID: 3TZR) of ligand-bound straight conformation of IRES subdomain IIa. Benzimidazole ligand is represented in red color. (C) Neighbouring base environment of U56 and U106 at bent conformation (PDB ID: 2NOK). Two neighbouring bases of U106 (G105 and G107) and two neighbouring bases of U56 (C55 and A57) are shown in red color. U106 and U56 are represented in magenta and green color, respectively. Mg^II and Mn^II are represented as cyan and yellow color sphere, respectively.

Figure 7

Fluorobenzofuran modified RNA ON 13 and 14 and their complementary RNA sequence 13c and 14c. They form respective modified IRES domain IIa duplexes, 13•13c and 14•14c where U56 and U106 residues were replaced with nucleoside 1.

Figure 8

Emission spectra and corresponding curve fits for the titration of subdomain IIa duplex 14•14c (0.5 μM) in sodium cacodylate buffer (10 mM, pH 6.5) with increasing concentration of Mg^II (A and B) and Mn^II (C and D) ions. Dashed lines represent the spectrum of duplexes in the absence of the metal ions.

Figure 9

¹⁹F NMR study of fluorobenzofuran-modified IRES subdomain IIa duplex 14•14c (50 μM) in 10 mM sodium cacodylate buffer (pH 6.5) in the absence and presence of 5 mM Mg^II.

Scheme 1

Synthesis of uridine analog 1 and its corresponding triphosphate 2. See SI for details.

Scheme 2

Synthesis of 5-fluorobenzofuran-modified uridine phosphoramidite 12. DMT = 4,4'-dimethoxytrityl, TBDMS = tert-butyldimethylsilyl, THF = tetrahydrofuran. See SI for details.