Synthesis of ¹⁸O-labeled RNA for application to kinetic studies and imaging

Abstract

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of (18)O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging (18)O atom into the phosphate group during the oxidation step of the synthetic cycle by using (18)O water as the oxygen donor. The (18)O label in the RNA was stable at pH 3-8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The (18)O/(16)O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of (18)O-labeled RNA, and this technique was used to determine the blood concentration of (18)O-labeled RNA after administration to mice. (18)O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.

Scheme 1.

¹⁸O-labeling of oligonucleotides during the oxidation step.

Figure 1.

LC-ESI-Q-Tof mass spectra of control and target siRNA. Green spectra indicate ¹⁶O-RNA, and red spectra indicate ¹⁸O-labeled RNA.

Figure 2.

RNAi activity of target siRNA. Each siRNA was transfected into A549 cells at the indicated concentrations by using Lipofectamine, and human GAPDH mRNA levels were determined by quantitative real-time PCR. Each value is the average of three independent experiments, and the error bars indicate the standard error.

Figure 3.

Determination of siRNA in the blood of mice after intravenous administration. (A) ¹⁸O/¹⁶O isotope ratio as a function of ¹⁸O-labeled control siRNA concentration. δ¹⁸O values were calculated from the results of IRMS, and the concentration of ¹⁸O-labeled control siRNA in the plasma was determined by its absorbance at 260 nm. Each value is the average of at least three independent experiments. (B) Blood concentration-time curve of control siRNA. The RNA concentrations were calculated from the δ¹⁸O values of the plasma samples. Each value is the average of at least three independent experiments.

Figure 4.

Isotope imaging of A549 cells transfected with unlabeled (A–D) or ¹⁸O-labeled control siRNA (E–H). Distribution of (A and E) ¹²C¹⁴N, (B and F) ¹⁶O and (C and G) ¹⁸O. (D and H) ¹⁸O/¹⁶O isotopographs. The isotopic abundance of ¹⁸O is indicated by the color scale.

Figure 5.

Histogram of the ¹⁸O isotope abundance of the foci in Figure 4H.