The i-motif structure is formed in cytosine-rich sequences, its building block being the cytosine·cytosine + base pair. This structure is particularly stable at pH values below physiological (∼7.4) and, because of that, it has not attracted as much biological interest as other non-canonical structures such as the G-quadruplex. Nowadays, the proposal of potential roles in vivo, as well as nanotechnological applications, has produced an increasing interest in its study. In this context, the present work provides an overall picture of the i-motif structure. Those aspects related to formation and stability, such as chemical modifications or the interaction with ligands, are discussed. Special attention has been made to the i-motif structures that could have a hypothetical role in vivo, such as those present near the promoter region of several oncogenes.

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.

An aptamer-capped mesoporous material for the selective and sensitive detection of α-thrombin in human plasma and serum has been prepared and characterised.

Selective elimination of metastatic stem cells (MetSCs) promises to block metastatic dissemination. Colorectal cancer (CRC) cells overexpressing CXCR4 display trafficking functions and metastasis‐initiating capacity. We assessed the antimetastatic activity of a nanoconjugate (T22‐GFP‐H6‐FdU) that selectively delivers Floxuridine to CXCR4⁺ cells. In contrast to free oligo‐FdU, intravenous T22‐GFP‐H6‐FdU selectively accumulates and internalizes in CXCR4⁺ cancer cells, triggering DNA damage and apoptosis, which leads to their selective elimination and to reduced tumor re‐initiation capacity. Repeated T22‐GFP‐H6‐FdU administration in cell line and patient‐derived CRC models blocks intravasation and completely prevents metastases development in 38–83% of mice, while showing CXCR4 expression‐dependent and site‐dependent reduction in foci number and size in liver, peritoneal, or lung metastases in the rest of mice, compared to free oligo‐FdU. T22‐GFP‐H6‐FdU induces also higher regression of established metastases than free oligo‐FdU, with negligible distribution or toxicity in normal tissues. This targeted drug delivery approach yields potent antimetastatic effect, through selective depletion of metastatic CXCR4⁺ cancer cells, and validates metastatic stem cells (MetSCs) as targets for clinical therapy. Synopsis Control of metastatic spread is an unmet need. This study reports on the therapeutic effect of a nanoconjugate (NC) that selectively delivers a drug to metastatic stem cells overexpressing surface CXCR4 receptor (CXCR4⁺ MetSCs) (Targeted drug delivery) after its intravenous injection in colorectal cancer (CRC) mouse models. Selective NC internalization, leading to a high genotoxic damage and elimination of CXCR4⁺CRC cells, in a CXCR4‐dependent way, both in vitro and in vivo. Reduction of tumor re‐initiation capacity and CXCR4⁺ tumor emboli intravasation in colonic peri‐tumoral vessels after NC treatment. Potent prevention of metastases, in cell line and patient‐derived orthotopic CRC mouse models, yielding a high percent of metastasis‐free mice after repeated‐dose therapy, as compared to the free drug. Metastatic sites with high CXCR4 expression show higher response to the NC and higher reduction in CXCR4⁺ cancer cell fraction at the end of treatment. Absence of accumulation or toxicity in normal tissues and high therapeutic index achieved by the NC, which exploits the high CXCR4 overexpression in target MetSCs in comparison to non‐tumor cells.

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.