RNA–RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA–RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA–RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA–RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA–RNA hybrids.

Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught more attention in recent times. Specifically, fusion of tumor cells with macrophages might lead to the development of metastasis by acquiring features such as genetic and epigenetic heterogeneity, chemotherapeutic resistance, and immune tolerance. In addition to the traditional FDA-approved definition of a CTC (CD45-, EpCAM+, cytokeratins 8+, 18+ or 19+, with a DAPI+ nucleus), an additional circulating cell population has been identified as being potential fusions cells, characterized by distinct, large, polymorphonuclear cancer-associated cells with a dual epithelial and macrophage/myeloid phenotype. Artificial fusion of tumor cells with macrophages leads to migratory, invasive, and metastatic phenotypes. Further studies might investigate whether these have a potential impact on the immune response towards the cancer. In this review, the background, evidence, and potential relevance of tumor cell fusions with macrophages is discussed, along with the potential role of intercellular connections in their formation. Such fusion cells could be a key component in cancer metastasis, and therefore, evolve as a diagnostic and therapeutic target in cancer precision medicine.

Aptamers able to bind efficiently cell-surface receptors differentially expressed in tumor and in healthy cells are emerging as powerful tools to perform targeted anticancer therapy. Here, we present a novel oligonucleotide chimera, composed by an RNA aptamer and a DNA decoy. Our assembly is able to (i) target tumor cells via an antitransferrin receptor RNA aptamer and (ii) perform selective codelivery of a chemotherapeutic drug (Doxorubicin) and of an inhibitor of a cell-survival factor, the nuclear factor κB decoy oligonucleotide. Both payloads are released under conditions found in endolysosomal compartments (low pH and reductive environment). Targeting and cytotoxicity of the oligonucleotidic chimera were assessed by confocal microscopy, cell viability, and Western blot analysis. These data indicated that the nuclear factor κB decoy does inhibit nuclear factor κB activity and ultimately leads to an increased therapeutic efficacy of Doxorubicin selectively in tumor cells.

Tumor cell‐surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single‐molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single‐molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live‐cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single‐molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.

Evasion of immune destruction is a major hallmark of cancer. Recent US Food and Drug Administration (FDA) approvals of various immunomodulating therapies underline the important role that reprogramming the immune system can play in combating this disease. However, a wide range of side effects still limit the therapeutic potential of immunomodulators, suggesting a need for more precise reagents with negligible off-target and on-target/off-tumor effects. Aptamers are single-chained oligonucleotides that bind their targets with high specificity and affinity owing to their three-dimensional (3D) structures, and they are one potential way to address this need. In particular, bispecific aptamers (bsApts) have been shown to induce artificial immune synapses that promote T cell activation and subsequent tumor cell lysis in various in vitro and in vivo pre-clinical models. We discuss these advances here, along with gaps in bsApt biology at both the cellular and resident tissue levels that should be addressed to accelerate their translation into the clinic. The broad application, minimal production cost, and relative lack of immunogenicity of bsApts give them some ideal qualities for manipulating the immune system. Building upon lessons from other novel therapies, bsApts could soon provide clinicians with an immunomodulating toolbox that is not only potent and efficacious but exercises a wide therapeutic index.

Significance: Neurotrophins (NTs) are homodimeric growth factors displaying fundamental roles in the nervous system. Their activity stems from binding and activation of 3 different receptor types in nervous cell membranes. The p75 NT receptor (p75 ^NTR ) was the first to be discovered in 1986; nevertheless, for the numerous structural and functional facets so far reported, its activation mechanisms have remained elusive. Here, we demonstrate that its pleiotropic functions are regulated by different redistributions of the receptors, which crucially depend on the available NT and on the involved subcellular compartment but are unrelated to its oligomerization state. Single-particle studies proved receptors to be monomers with a fast-diffusive behavior in the membrane with, at most, transient self-interactions on the millisecond time scale.