Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2'-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

Abstract

A long RNA oligomer, a 110mer with the sequence of a precursor-microRNA candidate, has been chemically synthesized in a single synthesizer run by means of standard automated phosphoramidite chemistry. The synthetic method involved the use of 2-cyanoethoxymethyl (CEM), a 2'-hydroxyl protecting group recently developed in our laboratory. We improved the methodology, introducing better coupling and capping conditions. The overall isolated yield of highly pure 110mer was 5.5%. Such a yield on a 1-mumol scale corresponds to 1 mg of product and emphasizes the practicality of the CEM method for synthesizing oligomers of more than 100 nt in sufficient quantity for biological research. We confirmed the identity of the 110mer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as HPLC, electrophoretic methods, and RNase-digestion experiments. The 110mer also showed sense-selective specific gene-silencing activity. As far as we know, this is the longest chemically synthesized RNA oligomer reported to date. Furthermore, the identity of the 110mer was confirmed by both physicochemical and biological methods.

Figure 1.

Synthesis of CEM-admities. (i) 2-cyanoethyl methylthiomethyl ether, molecular sieves 4A (MS 4A), NIS, CF₃SO₃H, THF, −45°C; (ii) DMSO, acetic anhydride, acetic acid; (iii) 3-hydroxypropionitrile, MS 4A, NIS, CF₃SO₃H, THF, −45°C; (iv) NH₄F, MeOH, 50°C or TEA·3HF, THF, 45°C; (v) 4,4′-dimethoxytrityl chloride, THF, pyridine, MS 4A, room temperature; (vi) diisopropylammonium tetrazolide, bis(N,N-diisopropylamino) cyanoethylphosphite, CH₃CN, 40°C.

Figure 2.

HPLC and electrophoretic analysis of purified 110mer RNA. (A) A PLRP-S 300 Å HPLC reverse-phase column was operated at a flow rate of 1 ml/min and maintained at a temperature of 80°C. The solvent system was buffers A and B, and the RNA was eluted with a linear gradient from 0 to 50% buffer B in 20 min. (B) A DNAPac PA100 HPLC anion-exchange column was operated at a flow rate of 1.5 ml/min and maintained at a temperature of 70°C. The solvent system was buffers C and D, and the RNA was eluted with a linear gradient from 5 to 50% buffer D in 20 min. (C) Capillary gel electrophoresis. (D) Polyacrylamide gel electrophoresis. The synthetic RNA was analyzed on a 5% polyacrylamide gel and stained with the cyanine dye SYBR Green II.

Figure 3.

Mass-spectrometric analysis of MazF cleavage products of the 110mer RNA. (A) Sequence of 110mer and the predicted cleavage products. (B) MALDI-TOF mass spectrum of MazF cleavage products.

Figure 4.

Mass-spectrometric analysis of RNase T1 cleavage products of the 110mer RNA. (A) MALDI-TOF mass spectrum of RNase T1 cleavage products. The inset shows an expanded view of the mass range 1860–1980. (B) Sequence of fragments produced by RNAse T1 digestion. The numbers shown represent the entry numbers of the fragments given in Table 2. Cleavage occurs on the 3′ side of G, and each number is placed at the G residue where cleavage occurs.

Figure 5.

HPLC analysis of enzymatically digested 110mer RNA. A Develosil ODS-UG-5 reverse-phase column (4.6 × 250 mm) was operated at a flow rate of 1 ml/min and maintained at a temperature of 40°C. The buffer was 50 mM KH₂PO₄, pH 3.0, with H₃PO₄/MeOH, 20:1 (v/v).

Figure 6.

Gene-silencing effect of chemically synthesized 110mer RNA. (A) Effect of 110mer RNA on expression of luciferase target gene. G3T-hi cells were transfected with pHOXB-Luc reporter plasmid and effector RNA (100 nM) and reporter assays performed 48 h after transfection. Each value shown is the average of three independent experiments, and standard deviations are indicated as error bars. (B) Comparison of sense/antisense suppression activity ratio for 110mer RNA. G3T-hi cells were transfected with pHOXB-Luc or pHOXB-Luc-antisense reporter plasmid and effector RNA (30 nM) and reporter assays performed 48 h after transfection. The results are presented as the ratio of the average of percent suppression of luciferase-containing sense and antisense target by 110mer RNA (pre-miRNA) or 22mer mature miRNA. (C) Sequences of the effector RNAs used. The underlined part of the 110mer pre-miR-196a-2 and the synthetic duplex 22mer miR-196a represent the sequence of the mature miRNA-196a.