Antisense Oligonucleotides as Tracers for PET Imaging

Oligonucleotides hybridize with high selectivity to RNA sequences allowing monitoring of gene expression or its inhibition in experimental and therapeutic applications. Custom-synthesized antisense oligonucleotides enable the design of molecular imaging technics needed for medical diagnostics. Molecular imaging technics allow visualization and quantification of molecular events in cellular contexts. In nuclear medicine, positron emission tomography (PET) and single-photon emission tomography (SPECT) are widely used in clinical therapeutic diagnostics, also called theranostics. PET-based imaging is fast enough to determine the pharmacokinetics of radiotracer uptake and distribution. PET typically utilizes radiolabeled molecules with positron-emitting nucleotides such as 15O, 13N, 11C, and 18F with relatively short half-lives. However, Gallium-68 and technetium-99m now also see increased use.

For the production of isotope-labeled probes, modified custom antisense oligonucleotides are provided with an amino group, a chelator, or any other functional group needed for labeling with the radioemitter located on either the 5’- or 3’-end ready for labeling with the radioemitter label.

In recent decades scientists developed several methods for labeling antisense oligonucleotides with positron-emitting isotopes. A selection of methods enables the labeling of antisense oligonucleotides with positron-emitting isotopes. Examples are positron-emitting isotopes 11C, 18F, or 76Br. Several molecular imaging trials with experimental animals have also been described. A recent example is the PET radioligand utilized for imaging 
Tau protein in the brain of patients with tauopathies.

Also, radiolabeled amino acid PET tracers targeting specific tumor-expressed receptors offer improved accuracy in defining the tumor-to-background contrast and in more particular treatments. Therefore, Gallium-68 (68Ga) has recently become an alternative positron emitter to the most commonly used 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG).

Pan et al., in 1998, utilized 5'-deoxy-5'-fluoro-O4-methylthymidine to develop radiofluorinated antisense oligodeoxynucleotide probes useful for PET.

In 1999, Kobori et al. imaged mRNAs in rat glioma tumors with antisense phosphorothioate oligodeoxynucleotides. The antisense oligonucleotides targeting the glial fibrillary acidic protein (GFAP) contained the positron emitter 11C as a label.

Roivainen et al., in 2004, reported that labeling oligonucleotides with 68Ga is a convenient approach for in vivo imaging and quantifying oligonucleotide biokinetics in living animals with PET.

Gallium-68 has a short half-life of 68 minutes. A germanium-68/gallium-68 generator allows its convenient production using a germanium-68/gallium-68 generator. These features enabled its increased clinical use in recent years.

Roivainen et al. labeled synthetic oligonucleotides with 68Ga and DOTA chelate conjugated to the oligonucleotides. Intravenously injected 68Ga-oligonucleotides of 17mer length generated high-quality PET images, allowing quantification of the biokinetics in major organs and tumors.

Liu et al., in 2007, showed that 99mTechnetium (99mTc)-labeled antisense oligonucleotide probes allowed imaging of human telomerase reverse transcriptase (hTERT) messenger RNA in malignant tumors. 99mTc is a metastable nuclear isomer of technetium-99. This isotope (Mw: 98.9063 Da) has a half-life of 6.0067 hours. The isotope enables a variety of imaging and diagnostic methods.

Cole et al., in 2014, showed that the short-lived fluorine-18 atom (t1/2 = 109.77 min) could be incorporated into molecular imaging probes late in the probe’s synthetic pathway. This approach enables the development of rapid and efficient late-stage fluorination methodologies.

Jacobsen et al., in 2015, reviewed labeling strategies and synthetic routes for Fluorine-18 radiochemistry. Typically, fluorine-18, with a nuclear characteristic of beta decay and a half-life of 109.7 minutes, is added to a functional group present at the reporter molecule (18F; 97% β+ decay, 109.7 min half-life, 635 keV).


Berger A. How does it work? Positron emission tomography. BMJ. 2003 Jun 28;326(7404):1449. [PMC]

Cole EL, Stewart MN, Littich R, Hoareau R, Scott PJ. Radiosyntheses using fluorine-18: the art and science of late stage fluorination. Curr Top Med Chem. 2014;14(7):875-900. [PMC]

Jacobson O, Kiesewetter DO, Chen X. Fluorine-18 radiochemistry, labeling strategies and synthetic routes. Bioconjug Chem. 2015 Jan 21;26(1):1-18. [

Kobori N, Imahori Y, Mineura K, Ueda S, Fujii R. Visualization of mRNA expression in CNS using 11C-labeled phosphorothioate oligodeoxynucleotide. Neuroreport. 1999 Sep 29;10(14):2971-4. [NeuroReport]

Liu M, Wang RF, Zhang CL, Yan P, Yu MM, Di LJ, Liu HJ, Guo FQ. Noninvasive imaging of human telomerase reverse transcriptase (hTERT) messenger RNA with 99mTc-radiolabeled antisense probes in malignant tumors. J Nucl Med. 2007 Dec;48(12):2028-36. [

Pan D, Gambhir SS, Toyokuni T, Iyer MR, Acharya N, Phelps ME, Barrio JR. Rapid synthesis of a 5'-fluorinated oligodeoxy-nucleotide: a model antisense probe for use in imaging with positron emission tomography (PET). Bioorg Med Chem Lett. 1998 Jun 2;8(11):1317-20. [

Roivainen A, Tolvanen T, Salomäki S, Lendvai G, Velikyan I, Numminen P, Välilä M, Sipilä H, Bergström M, Härkönen P, Lönnberg H, Långström B. 68Ga-labeled oligonucleotides for in vivo imaging with PET. J Nucl Med. 2004 Feb;45(2):347-55. [