Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

11C-radiolabeled aptamer for imaging of tumors and metastases using positron emission tomography- computed tomography.

Identification of primary tumors and metastasis sites is an essential step in cancer diagnostics and the following treatment. Positron emission tomography-computed tomography (PET/CT) is one of the most reliable methods for scanning the whole organism for malignancies. In this work, we synthesized an 11C-labeled oligonucleotide primer and hybridized it to an anti-cancer DNA aptamer. The 11C-aptamer was applied for in vivo imaging of Ehrlich ascites carcinoma and its metastases in mice using PET/CT. The imaging experiments with the 11C-aptamer determined very small primary and secondary tumors of 3 mm2 and less. We also compared 11C imaging with the standard radiotracer, 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG), and found better selectivity of the 11C-aptamer to metastatic lesions in the metabolically active organs than 18F-FDG. 11C radionuclide with an ultra-short (20.38 min) half-life is considered safest for PET/CT imaging and does not cause false-positive results in heart imaging. Its combination with aptamers gives us high-specificity and high-contrast imaging of cancer cells and can be applied for PET/CT-guided drug delivery in cancer therapies.

The role of SAXS and molecular simulations in 3D structure elucidation of a DNA aptamer against lung cancer.

Aptamers are short, single-stranded DNA or RNA oligonucleotide molecules that function as synthetic analogs of antibodies and bind to a target molecule with high specificity. Aptamer affinity entirely depends on its tertiary structure and charge distribution. Therefore, length and structure optimization are essential for increasing aptamer specificity and affinity. Here, we present a general optimization procedure for finding the most populated atomistic structures of DNA aptamers. Based on the existed aptamer LC-18 for lung adenocarcinoma, a new truncated LC-18 (LC-18t) aptamer LC-18t was developed. A three-dimensional (3D) shape of LC-18t was reported based on small-angle X-ray scattering (SAXS) experiments and molecular modeling by fragment molecular orbital or molecular dynamic methods. Molecular simulations revealed an ensemble of possible aptamer conformations in solution that were in close agreement with measured SAXS data. The aptamer LC-18t had stronger binding to cancerous cells in lung tumor tissues and shared the binding site with the original larger aptamer. The suggested approach reveals 3D shapes of aptamers and helps in designing better affinity probes.