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Synthesis of ¹?O-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.

SUBMITTER: Hamasaki T

PROVIDER: S-EPMC3695515 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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

Hamasaki Tomohiro T Matsumoto Takahiro T Sakamoto Naoya N Shimahara Akiko A Kato Shiori S Yoshitake Ayumi A Utsunomiya Ayumi A Yurimoto Hisayoshi H Gabazza Esteban C EC Ohgi Tadaaki T

Nucleic acids research 20130430 12

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 int ...[more]

PMID: 23632164

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Project description:RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (~25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific (13)C/(15)N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling, and selective-biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example, total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10%) that drop precipitously for oligonucleotides larger than 50 nt. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1' and C5' and the pyrimidine nucleobase C2, C4, C5, or C6. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places (13)C/(15)N labels in the pyrimidine nucleobase (either (15)N1, (15)N3, (13)C2, (13)C4, (13)C5, or (13)C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al., 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full-length riboswitches larger than the ~25 nt median size of RNA NMR structures found in the PDB.

Project description:Selective modulation of metabotropic glutamate receptor 2 (mGlu2) represents a novel therapeutic approach for treating brain disorders, including schizophrenia, depression, Parkinson's disease (PD), Alzheimer's disease (AD), drug abuse and addiction. Imaging mGlu2 using positron emission tomography (PET) would allow for in vivo quantification under physiological and pathological conditions and facilitate drug discovery by enabling target engagement studies. In this paper, we aimed to develop a novel specific radioligand derived from negative allosteric modulators (NAMs) for PET imaging of mGlu2. Methods. A focused small molecule library of mGlu2 NAMs with tetrahydro naphthyridine scaffold was synthesized for pharmacology and physicochemical evaluation. GIRK dose-response assays and CNS panel binding selectivity assays were performed to study the affinity and selectivity of mGlu2 NAMs, among which compounds 14a and 14b were selected as PET ligand candidates. Autoradiography in SD rat brain sections was used to confirm the in vitro binding specificity and selectivity of [11C]14a and [11C]14b towards mGlu2. In vivo binding specificity was then studied by PET imaging. Whole body biodistribution study and radiometabolite analysis were conducted to demonstrate the pharmacokinetic properties of [11C]14b as most promising PET mGlu2 PET ligand. Results. mGlu2 NAMs 14a-14g were synthesized in 14%-20% yields in five steps. NAMs 14a and 14b were selected to be the most promising ligands due to their high affinity in GIRK dose-response assays. [11C]14a and [11C]14b displayed similar heterogeneous distribution by autoradiography, consistent with mGlu2 expression in the brain. While PET imaging study showed good brain permeability for both tracers, compound [11C]14b demonstrated superior binding specificity compared to [11C]14a. Further radiometabolite analysis of [11C]14b showed excellent stability in the brain. Conclusions. Compound 14b exhibited high affinity and excellent subtype selectivity, which was then evaluated by in vitro autoradiography and in vivo PET imaging study after labeling with carbon-11. Ligand [11C]14b, which we named [11C]MG2-1904, demonstrated high brain uptake and excellent in vitro/in vivo specific binding towards mGlu2 with high metabolic stability in the brain. As proof-of-concept, our preliminary work demonstrated a successful example of visualizing mGlu2in vivo derived from NAMs, which represents a promising chemotype for further development and optimization aimed for clinical translation.

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Synthesis of ¹?O-labeled RNA for application to kinetic studies and imaging.

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

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