Chemicals.
All materials and solvents were obtained commercially and used without further purification unless special statement. Mature miR-21 sequence, the complementary strand 5′-UAGCUUAUCAGACUGAUGUUGA-3′, was purchased from Suzhou GenePharma Co., Ltd. F-miR-21 oligomers, the complementary strand 5′-FAM-UAGCUUAUCAGAC UGAUGUUGA-BHQ-1-3′, was also purchased from Suzhou GenePharma Co., Ltd. Both isomers Λ/Δ-[Ru(bpy)2(p-TEPIP)](ClO4)2 (Λ/Δ-1 ) were synthesized as previously described in the literature [59]. The detailed characterization data are listed in Supporting information (Figures S1–S4), and the purity of Λ/Δ-1 was higher than 95% as tested by HPLC (Figure S5).
Quantitative reverse transcription-PCR assay for miR-21 expression.
Quantitative PCR (Q-PCR) analysis of miR-21 transcript expression profiles of both isomers at various confluences were measured using the AB 7900HT Real-Time PCR system as previously described [60] with some modifications. All conditions of this experiment were similar to Bcl-2, except miR-21 was replaced. The primers were as follows: miR-21 Forward, 5′-ACACTCCAGCTGG GTAGCTTATCAGACTGA-3′; Reverse, 5′-GTGTCG TGGAGTCGGCAATTC-3′; U6 Forward, 5′-GTGCTC GCTTCGGCAGCACATATAC-3′; Reverse, 5′ AAAAA TATGGAACGCTTCACGAATTTG-3′.
Electronic absorption measurements.
Electronic absorption spectra of the ruthenium complexes (10 μM) interacted with increasing concentration of miR21 (0, 0.0667, 0.1334, 0.2 ...... 0.667 μM) were performed on a Shimadzu UV-2550 spectrophotometer at room temperature. [61]. The absorption spectrum was recorded after mixing the ruthenium(II) complex-miR-21 solution for 5 min. The addition of miR21 repeated some times until little changes were observed in the spectra, suggesting that the binding saturation was achieved.
Fluorescence measurements.
Fluorescence experiments were carried out by inreasing miR-21 solution to the ruthenium(II) complexes. The fluorescence of both ruthenium complexes were excited at 340 nm, and the emission curve was observed from 500-700 nm. The fluorescence spectrum was recorded after mixing the ruthenium(II) complex-miR-21 solution for 5 min. The addition of miR21 repeated some times until little changes were observed in the spectra, suggesting that the binding saturation was achieved. Due to the dilution after each titration experiment, the concentration of the ruthenium(II) complex only slightly changed.
Fluorescence resonance energy transfer melting point assay.
The FRET melting point assay was performed to clarify the affinity of chiral ruthenium(II) complexes to bind miR-21. Fluorescence melting curves were measured by a Bio-Rad real-time PCR detection system using a total reaction excitation at 470 nm constant temperature being maintained for 30 s prior to each reading to ensure a stable value [62].
Circular dichroism spectra measurements.
Circular dichroism spectra were obtained using a Jasco J810 spectropolarimeter [63]. During the titration, a 2 μL aliquot of buffered miR-21 solution was added to each cuvette of drugs, and the solutions were mixed by repeated inversion. After the solutions were mixed for ~5 min, the CD spectra were recorded. For each sample, CD experiments were measured at least three times at room temperature by using a quartz cell with a path length of 1 cm. The spectra were collected at wavelengths of 200–600 nm and with a scanning speed of 50 nm/min [64]. The instrument was flushed continuously with pure evaporated nitrogen throughout the experiment.