99mTc-Mercaptoacetyl-Glu-Glu-aptamer specific for tenascin-C

Excerpt

Tenascin-C is a large, adhesive, extracellular matrix (ECM) glycoprotein (>10³ kDa) (1) with numerous alterative names such as myotendinous antigen, glioma mesenchymal ECM protein (GMEM), cytotactin, J1-200/220, hexabrachion, and neuronectin (2). Tenascin-C contains six disulphide-linked subunits spread as six arms from a central core (3). Each subunit possesses multiple-function domains, including a cysteine-rich N-terminal domain associated with self-oligomerization, followed by epidermal growth factor (EGF)-like repeats, fibronectin (FN) type III-like repeats, and a fibrinogen-like globular domain at the C-terminus. Consequently, tenascin-C can bind to different cell-surface receptors such as integrins and ECM components (FN). Tenascin-C is found to express specifically in damaged tissues, vascular diseases, and a majority of malignant solid tumors (4).

Aptamers (from the Latin aptus, to fit, and the Greek meros, part or region) are single-stranded or double-stranded oligonucleotides (DNA or RNA) that are modified to bind a variety of targets with high binding affinity and specificity (5). Aptamers typically consist of 20–50 nucleotides (8–15 kDa) with dissociation constants in the range of 10 pM to 10 nM (6). Unlike linear oligonucleotides, which contain genetic information or antisense oligonucleotides that interrupt the transcription of genetic information, aptamers are globular molecules with a shape similar to tRNA and bind to target proteins specifically (7). The modification of the oligonucleotides is carried out through the systematic evolution of ligands by exponential enrichment (SELEX) method, in which specific aptamers are generated against desired small proteins, cells, and tissues (8). In general, aptamers are capable of distinguishing between closely related members of a protein family and between different functional or conformational states of the same protein (6). Aptamers have been used in various clinical trials as alternate therapeutics in cancers (6). For in vivo applications, aptamers can be modified chemically against degradation caused by exonucleases and endonucleases (9).

TTA1 is a 39mer oligonucleotide (13.4 kDa) that binds specifically to human tenascin-C with high affinity (dissociation constant, 5 × 10^-9 M) (9). To enhance its resistance to blood and tissue nucleases, the structure of TTA1 is modified in several locations (9). All pyrimidine ribonucleotides are replaced with 2’-deoxy-2’-fluoronucleolytic nucleotides, 14 of the 19 purine ribonucleotides are replaced with 2’-deoxy-2’-OMe nucleotides, the 3’ end is blocked with a 3’-3’-thymidine cap for exonuclease protection, a (CH₂CH₂O)₆ spacer is connected between oligonucleotide chains, and a 5’ hexyl-aminolinker is added for bioconjugation of imaging probes. Connecting this aminolinker with radionuclide chelators, technetium-labeled mercaptoacetyl-Glu-Glu (^99mTc-MAG₂), yields the molecular imaging agent ^99mTc-mercaptoacetyl-Glu-Glu-aptamer specific for tenascin-C (^99mTc-TTA1) (8). ^99mTc is a common radioactive label used in single-photon emission computed tomography (SPECT) imaging, and it has a 0.90 gamma branch factor, a 6-h half-life at 140 keV, and a low isotope cost ($0.21/mCi) (10). ^99mTc-TTA1 appears to be a promising candidate for imaging of tumors expressing tenascin-C (5).