Molecular imaging with nucleic acid aptamers

Abstract

With many desirable properties such as ease of synthesis, small size, lack of immunogenicity, and versatile chemistry, aptamers represent a class of targeting ligands that possess tremendous potential in molecular imaging applications. Non-invasive imaging of various disease markers with aptamer-based probes has many potential clinical applications such as lesion detection, patient stratification, treatment monitoring, etc. In this review, we will summarize the current status of molecular imaging with aptamer-based probes. First, fluorescence imaging will be described which include both direct targeting and activatable probes. Next, we discuss molecular magnetic resonance imaging and targeted ultrasound investigations using aptamer-based agents. Radionuclide-based imaging techniques (single-photon emission computed tomography and positron emission tomography) will be summarized as well. In addition, aptamers have also been labeled with various tags for computed tomography, surface plasmon resonance, dark-field light scattering microscopy, transmission electron microscopy, and surface-enhanced Raman spectroscopy imaging. Among all molecular imaging modalities, no single modality is perfect and sufficient to obtain all the necessary information for a particular question. Thus, a multimodality probe has also been constructed for concurrent fluorescence, gamma camera, and magnetic resonance imaging in vivo. Although the future of aptamer-based molecular imaging is becoming increasingly bright and many proof-of-principle studies have already been reported, much future effort needs to be directed towards the development of clinically translatable aptamer-based imaging agents which will eventually benefit patients.

Fig. 1

A QD-Apt-Dox conjugate. A. QD-Apt-Dox is initially “off” as the fluorescence of QD is transferred to Dox and the fluorescence of Dox is quenched by the aptamer, both by fluorescence resonance energy transfer. B. Once QD-Apt-Dox is inside cancer cells, Dox is gradually released from the conjugate and the fluorescence of QD is recovered. C. Microscopy images of PSMA-positive cells after incubation with QD-Apt-Dox. QD and Dox are shown in green and red, respectively. Scale bar: 20 µm. Adapted from [33,126].

Fig. 2

Fluorescence imaging with an aptamer-based activatable probe. A. A schematic representation of the imaging strategy based on cell membrane protein-triggered conformation alteration. The probe is consisted of three fragments: a cancer-targeted aptamer sequence (A-strand), a poly-T linker (T-strand), and a short DNA sequence (C-strand) complementary to a part of the A-strand, with a fluorophore (F) and a quencher (Q) attached at either terminus. When no target is present, the probe is hairpin-structured with quenched fluorescence. When the probe binds to targeted cancer cells, its conformation is altered which results in enhanced fluorescence signal. B. Serial in vivo fluorescence imaging of tumor-bearing mice after intravenous injection of the activatable probe, a control probe, or an “always-on” probe. The circles in the images denote the tumor sites. Adapted from [59].

Fig. 3

Molecular MRI with an aptamer-based probe. A. The iron oxide nanoparticles (red spheres) were modified with DNA aptamers that binds to fibrinogen-recognition exosite of thrombin (blue lines) or heparin-binding exosite of thrombin (green lines). Addition of thrombin consisting of both fibrinogen (blue donuts) and heparin (green donuts) exosites results in nanoparticle aggregation, which leads to reduced T2 relaxation time. B. T2-weighted magnetic resonance images of 1:1 nanoparticle mixture in the presence of various concentrations of thrombin (in the unit of nanomolar). Adapted from [66].

Fig. 4

SPECT imaging with a ^99mTc-labeled aptamer. A. Structure of the aptamer. Pyrimidines are 2’-F and purines are 2’-OMe, except at arrowheads, where purines remain 2’-OH. The non-binding control aptamer has 5-nucleotide internal deletion. B. Serial gamma camera imaging of tumors (arrowheads) after intravenous injection of ^99mTc-labeled aptamers. Adapted from [86].

Fig. 5

Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance (SPR) imaging. A. In step a, the target protein binds to the surface-immobilized aptamer. In step b, a horseradish peroxidase (HRP)-conjugated antibody to the target protein is introduced to create a surface aptamer-protein-antibody sandwich structure. In step c, this surface is exposed to the substrate 3,3’,5,5’-tetramethylbenzidine (TMB), which reacts with HRP to form a dark blue precipitate on the array elements containing the complex, detectable by SPR imaging. B. Pattern of the three-component RNA microarray for detection, each color represents the use of a RNA aptamer against a different target. C. A SPR difference image obtained for R₂. Adapted from [109].

Fig. 6

Multimodality imaging with an aptamer-based probe. A. A schematic illustration of the probe MFR-AS1411. B. Serial gamma camera imaging of tumor-bearing mice after intravenous injection of MFR-AS1411 or a control probe which contains a mutated aptamer. Arrows indicate tumors. C. Magnetic resonance images of tumor-bearing mice before and after injection of MFR-AS1411. Dark signal intensities at tumor sites (arrowheads) were detected in mice injected with MFR-AS14110. D. Ex vivo fluorescence imaging of major organs. Dashed ovals indicate the tumors. Adapted from [113].