Oligonucleotides in PET imaging

Nucleotides are the build blocks of DNA and RNA, consisting of a nucleobase (adenine, cytosine, guanine, thymine, or uracil), a 5-carbon sugar, and a phosphate group. Oligonucleotide can hybridize to another chain if their sequences can form G☰C and A⚌T or A⚌U pairs. Thus, oligonucleotides can be used as ligands for DNA and RNA (antisense oligonucleotides). Oligonucleotides can also be designed to specifically bind to other molecular targets (aptamer oligonucleotides). Techniques for generic labelling and protection from nucleases are available, but directing oligonucleotides into the cell cytoplasm and nucleus while still maintaining their functionality is still challenging.

In blood plasma, oligonucleotides are bound to albumin (Srinivasan et al., 1995), which is common to negatively charged molecules. Oligonucleotides tend to bind to other proteins, too, and while this may not be a problem for medicinal use of oligonucleotide, it reduces image quality by directly increasing the nonspecific binding and indirectly by limiting glomerular filtration and urinary excretion, increasing the halflife of the radiopharmaceutical in the plasma.

Antisense oligonucleotides

Antisense single-stranded oligonucleotides (ASOs) bind to its target RNA, and inhibit its translation (Seth et al., 2019). Labelled ASOs could be used for imaging, but the number of mRNAs copies in a cell may be too low for detection in vivo. Antisense imaging may therefore be most successful in detecting viral or bacterial mRNAs that are abundant in infected tissue. The secondary structures of RNAs (hairpins, stem-loops) may prevent or slow down the hybrid formation with the antisense oligonucleotide.

Chemical modification of ASOs

Several chemical modifications have been introduced to increase the stability and/or binding affinity of ASOs (Lennox & Behlke, 2011).

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic molecules which have standard nucleic acid bases, but those bases are bound to non-ribose methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates (Summerton & Weller, 1997). PMOs are more stable in vivo than RNA or DNA oligonucleotides. In contrast to phosphate groups, the phosphorodiamidate groups are uncharged in physiological pH, which leads to faster and tighter duplex formation with their complementary DNA or RNA sequence.

In phosphorothioate (PS) modification a sulphur atom is substituted for a non-bridging oxygen in the phosphate backbone. This reduces the ability of many nucleases to degrade the bond, but also reduces the binding affinity of PS-modified oligonucleotides to their targets.

Aptamer oligonucleotides

DNA and RNA aptamers (usually 20-100 bases) are usually single-stranded, and can form 3D-structures, enabling them to bind to a specific target (aptatope); targets can be anything from small ions such as Zn2+ to viruses and bacteria. Libraries of oligonucleotides can be synthesized randomly and simultaneously screened for their binding affinity to a target molecule. This procedure is easier and faster than the methods for production of monoclonal antibodies, and it works with targets that are toxic or non-immunogenic. Aptamers are an order of magnitude smaller than antibodies (10-20 kDa versus 150 kDa), and therefore have faster endothelial passage and clearance. Aptamers, like antibodies, are usually targeted against molecules that are abundant enough for successful detection with the excellent sensitivity of modern PET devices.

Escort aptamers can be used for targeted delivery of drugs, and diagnostic or therapeutic radionuclides.

Riboswitches are naturally occurring aptamers, mainly found in the untranslated regions of mRNA molecules; when bound to their target molecule, the 3D structural change in mRNA affects its translational activity.

Promising aptamer radiopharmaceuticals have been developed for example for tumour PET imaging, targeting EGFR (HER1) (Cheng et al., 2019) and PTK7 (Jacobson et al., 2015).


MicroRNAs (miRNAs) are endogenous single-stranded non-coding RNAs consisting of 16-23 nucleotides. MicroRNAs regulate gene expression by inhibiting the post-transcriptional process: miRNA pairs with messenger RNA (mRNA) to its untranslated regions, which leads to formation of miRNA-induced silencing complex (miRISC) that includes ribonucleases. If the sequences of miRNA and mRNA do not match well, the miRISC merely inhibits the translation of that mRNA, but when the match is good, then miRISC starts RNA degradation process (RNA interference, RNAi) (Hernandez et al., 2013). Thousands of miRNAs have been identified. Certain miRNA (for instance miR-21) are often over-expressed or down-regulated in certain tumour cells. miR-15b is important for tissue remodelling, especially in the bones. Short interfering RNAs (siRNAs) function similarly, but are exogenous double-stranded RNAs. Pre-miRNAs are transcribed in the nucleus, and digested into mature miRNAs in the cytoplasm (Mäkilä et al., 2019). In blood, miRNAs are mainly located in erythrocytes, which are involved in endothelial function and cardiovascular diseases (Kontidou et al., 2023).

miRNAs and siRNAs are being tested as therapeutic agents, but their short in vivo halflife, low cellular uptake (via endocytosis), and off-target effects remain a problem. Pharmacokinetics, biodistribution, and specific tissue uptake of positron-emitting radionuclide labelled miRNAs with PET (Mäkilä et al., 2019).

Spherical nucleic acids

Spherical nucleic acids (SNAs) consist of nanoparticle core functionalized with a shell of oriented oligonucleotides (Cutler et al., 2012; Kapadia et al., 2018). SNAs can penetrate the skin and cross the blood-brain barrier. SNAs can enter most cells via active scavenging processes (Rosi et al., 2006; Choi et al., 2013). In contrast to most nanoparticles, SNAs do not trigger unwanted immune response (Massich et al., 2010), but phagocytes can still actively scavenge SNAs, which can lead to high accumulation in the spleen and liver. Mature red blood cells do not scavenge SNAs. High-valency polydisperse SNAs based on gold and lipid nanoparticles can have very high affinity towards their targets but also towards scavenger receptors, while low-valency molecular spherical nucleic acids (MSNAs) assembled on a [60]fullerene core can stay longer in circulation (Äärelä et al., 2023).

DNA- and RNA-SNAs are degraded by serum nucleases, but the stability can be increased by increasing nucleic acid density and other structural modifications (Barnaby et al., 2016). PMO and PS modification of ASOs increases the stability and circulation time considerably.

In blood, SNAs interact with plasma proteins, and the formation of protein corona can influence the targeting and pharmacokinetics of the SNA. However, SNAs can be tailored to have an active protein coronae, improving targeting of specific cell populations (Zhang et al., 2019). Large particle size prevents renal clearance.

Labelling SNAs for PET or SPECT imaging requires using radionuclides with long half-lives because of the slow pharmacokinetics. Alternatively, short-lived radionuclides can be used with pretargeted imaging approach. For instance, TCO-functionalized SNAs can be labelled in vivo with [18F]FDG-Tz, produced from widely available PET tracer [18F]FDG (Auchynnikava et al., 2023).

See also:


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Updated at: 2024-01-15
Created at: 2017-10-28
Written by: Vesa Oikonen