Synthesis and Enzymatic Incorporation of a Responsive Ribonucleoside Probe That Enables Quantitative Detection of Metallo-Base Pairs

Synthesis of a highly responsive fluorescent ribonucleoside analogue based on a 5-methoxybenzofuran uracil core, enzymatic incorporation of its triphosphate substrate into RNA transcripts, and its utility in the specific detection and estimation of Hg2+-ion-mediated metallo-base pair formation in DNA–RNA and RNA–RNA duplexes are described.

S4 Figure S1. Relative quantum yield versus E T (30) plot for nucleoside 1 and other heterocycleconjugated nucleoside probes.

5-(5-methoxybenzofuran)-uridine triphosphate (2):
To a solution of 5-(5methoxybenzofuran)-uridine (1) Where s is the standard, x is the modified nucleoside, A is the absorbance at excitation wavelength, F is the area under the emission curve, n is the refractive index of the solvent, and Ф F is the quantum yield. Quantum yield of 2-aminopurine in water is 0.68. μCi α-32 P ATP and 3 U/μL (total 60 units) T7 RNA polymerase in a total 20 μL reaction volume. After 3.5 h, the reaction was quenched using 20 μL of loading buffer (7 M urea in 10 S6 mM Tris-HCl, 100 mM EDTA, 0.05% bromophenol blue, pH 8). The samples were heated at 75 °C for 3 min and then cooled on an ice bath. The samples (4 μL) were loaded on a sequencing 18% denaturing polyacrylamide gel and were electrophoresed at a constant power (11 W) for nearly 4 h. The bands corresponding to the radioactive products were imaged using an X-ray film. The relative transcription efficiency was calculated using GeneTools software from Syngene. The % of incorporation of modified UTP 2 into RNA transcripts was determined considering the transcription efficiency in presence of all natural NTPs as 100 %.
All reactions were performed in duplicate and the errors in yields were ≤ 4%. See Figure S2. The transcription reaction was performed at 37 °C for 12 h. The reaction volume was reduced to almost one third of the total volume using speed vac. 40 µL loading buffer (10 mM Tris HCl, 7 M urea, 100 mM EDTA, pH 8.0) was added to the residual mixture and loaded onto to S7 the 20% polyacrylamide gel and run at 25 W constant power for 6 h. Gel was UV shadowed, the band corresponding to full-length transcript was cut and transferred to the Poly-Prep column (Bio-Rad). The gel pieces were crushed; the transcript was extracted with 0.5 M ammonium acetate for 12 h and desalted using Sep-Pak classic C18 cartridges (Waters).
The purity of the transcript was examined by HPLC and the transcript was characterized by MALDI TOF mass analysis (see Figure S3).

MALDI-TOF mass analysis of transcript 4:
A mixture of 2 µL of the transcript 4 (~200 µM), 4 µL of a 9:1 solution of 3-hydroxypicolinic acid and ammonium citrate buffer (100 mM, pH 9) and 2 µL of an internal DNA standard (100 µM) was desalted using an ionexchange resin (Dowex 50W-X8, 100-200 mesh, ammonium form). The sample was then spotted on a MALDI plate, air dried and subjected to mass analysis. The spectrum was calibrated relative to the internal DNA standard (see Figure S3).  Figure S4). Further to characterize the ribonucleosides in digest, the fraction corresponding to the individual ribonucleoside was collected and analyzed by mass spectroscopy (Table S1). Figure S4. HPLC chromatogram of (A) Natural ribonucleosides rC, rU, rG, rA and nucleoside 1 mix. and (B) ribonucleosides obtained from enzymatic digestion of transcript 4. See section 8 for more details.   CD measurements were performed in duplicate and all spectra were corrected using an appropriate blank solution in the absence of ONs.   12. Thermal melting analysis of ON duplexes. ON duplexes (5 µM) were formed in sodium cacodylate buffer (10 mM, pH 7.0) containing 500 mM NaNO 3 as mentioned earlier, either in presence or absence of 1 equivalent Hg 2+ ions (5 µM). Thermal melting analysis was performed using Cary 300 Bio UV-Vis spectrophotometer. Temperature was increased from 18 °C to 90 °C at 1 °C/min and the absorbance was measured every 1 °C interval at 260 nm.  containing 500 mM NaNO 3 similarly like steady-state fluorescence sample. After recording the 1 H NMR of the duplexes without Hg 2+ ions, the concentration of Hg 2+ in the sample was increased gradually by adding higher concentrations (2.5 mM) of Hg 2+ ions such that the final concentration of the metal salt was 25 µM (0.5 equiv) and 50 µM (1 equiv). After addition of Hg 2+ , samples were annealed as mentioned above. Figure S11. Partial 1 H NMR spectra of duplexes 4•8 with dT-1 mismatch (50 µM) and 4•10 with U-1 mismatch in the absence and presence of 0.5 equivalent (25 µM) and 1.0 equivalent (50 µM) Hg 2+ ions. Spectra were recorded at 20 ºC in sodium cacodylate buffer (10 mM, pH 7.0) containing 500 mM NaNO 3 . Imino proton signals corresponding to mispair is shown here, which disappears upon metal ion coordination. S8 S15 14. NMR Spectra 1 H NMR of 1b (400 MHz, CDCl 3 containing 0.03% (v/v) TMS) 13 C NMR of 1b (100 MHz, CDCl 3 )