A novel tetrapeptide for chelator-free radiolabeling in optimized preparation of ^99mTc-radiolabeled oligonucleotides

Antisense imaging uses radionuclide labeled antisense oligonucleotides to hybridize with nucleic acids in vivo, display the expression of target genes, and directly quantify biological processes at the cellular and subcellular levels. The anti-miRNA oligonucleotides (AMOs) are a series of single-stranded DNA oligonucleotides that are widely used in gene imaging and gene therapy. However, due to the negative charge and high molecular weight, the permeability through the membrane of AMOs is generally low so that most AMOs cannot enter the cells. Based on the ^99mTc-labeled AMOs imaging in previous studies, this study developed a novel tetrapeptide Glycine-Alanine-Glycine-Lysine (Gly-Ala-Gly-Lys, GAGK) for one-step labeling AMO with ^99mTc. The labeling conditions were optimized by changing the number of stannous ions, the reaction time, and the temperature, respectively. The labeled products were identified by gel electrophoresis and their serum stability was evaluated. The optimal labeling condition in this study was using 1 mg/mL SnCl₂·2H₂O and heating for 30 min at 100°C. Gel electrophoresis confirmed the verification of successful labeling of ^99mTc-GAGK-AMO. After being incubated with human fresh serum for 12 h, ^99mTc-GAGK-AMO showed good stability and no obvious degradation. Therefore, this labeling method has high labeling efficiency and stable labeling, which provides an effective method for the application of miRNA-targeted imaging.