The thrombin binding aptamer (TBA) is a well characterized chair-like, antiparallel quadruplex structure that binds specifically to thrombin at nanomolar concentrations and therefore it has interesting anticoagulant properties. In this article we review the research involved in the development of new TBA derivatives with improved anticoagulant properties as well as the use of the TBA as a model compound for the study of quadruplex structures. Specifically, we describe the impact of modified nucleosides and non-natural backbones in the guanine tetrads or in the loops and the introduction of pendant groups at the 3’ or 5’-ends. The modified oligonucleotides are shown to be excellent tools for the understanding of the molecular structure of the TBA and its folding properties. Finally, we review the use of the TBA-Thrombin recognition system for the development of analytical tools based on the TBA folding.

Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs, are considered one of the most successful agents in the treatment of colorectal cancer, yet poor specificity and tumor cell resistance remain the major limiting bottlenecks. Here, we exploited for the first time the ability of two DNA nanoscaffolds, a DNA tetrahedron (Td) and rectangle DNA origami, to incorporate 5-fluoro-2′-deoxyuridine (FdU_n) oligomers. In addition, cholesterol moieties were synthetically attached to Td and DNA origami staples to enhance cellular uptake. DNA nanostructures functionalized with FdU_n exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-FU and FdU, especially having cholesterol as an internalization helper. The cholesterol content mostly correlates with the increase of the FdU_n nanostructure cytotoxicity. DNA nanoscaffolds bearing FdU_n were able to circumvent the low sensitivity of colorectal cancer cells towards 5-FU. Both DNA nanostructures attained a comparable cytotoxic effect yet Td displays higher antiproliferative action. The ability to reduce the proliferation of cancer cells is mainly related to the concentration of DNA nanostructures. The present work suggests that self-assembled DNA nanoparticles are privileged vehicles for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment.