Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

Mixtures of interacting compounds produced by plants may provide important combination therapies that simultaneously affect multiple pharmacological targets and provide clinical efficacy beyond the reach of single compound-based drugs. Developing innovative scientific methods for discovery, validation, characterization and standardization of these multicomponent botanical therapeutics is essential to their acceptance into mainstream medicine.

Plants have been used as a source of medicine throughout history and continue to serve as the basis for many pharmaceuticals used today. Although the modern pharmaceutical industry was born from botanical medicine, synthetic approaches to drug discovery have become standard. However, this modern approach has led to a decline in new drug development in recent years and a growing market for botanical therapeutics that are currently available as dietary supplements, drugs, or botanical drugs. Most botanical therapeutics are derived from medicinal plants that have been cultivated for increased yields of bioactive components. The phytochemical composition of many plants has changed over time, with domestication of agricultural crops resulting in the enhanced content of some bioactive compounds and diminished content of others. Plants continue to serve as a valuable source of therapeutic compounds because of their vast biosynthetic capacity. A primary advantage of botanicals is their complex composition consisting of collections of related compounds having multiple activities that interact for a greater total activity.

Paraspeckles are nuclear bodies that regulate multiple aspects of gene expression. The long non-coding RNA (lncRNA) NEAT1 is essential for paraspeckle formation. NEAT1 has a highly ordered spatial organization within the paraspeckle, such that its 5′ and 3′ ends localize on the periphery of paraspeckle, while central sequences of NEAT1 are found within the paraspeckle core. As such, the structure of NEAT1 RNA may be important as a scaffold for the paraspeckle. In this study, we used SHAPE probing and computational analyses to investigate the secondary structure of human and mouse NEAT1. We propose a secondary structural model of the shorter (3,735 nt) isoform hNEAT1_S, in which the RNA folds into four separate domains. The secondary structures of mouse and human NEAT1 are largely different, with the exception of several short regions that have high structural similarity. Long-range base-pairing interactions between the 5′ and 3′ ends of the long isoform NEAT1 (NEAT1_L) were predicted computationally and verified using an in vitro RNA–RNA interaction assay. These results suggest that the conserved role of NEAT1 as a paraspeckle scaffold does not require extensively conserved RNA secondary structure and that long-range interactions among NEAT1 transcripts may have an important architectural function in paraspeckle formation.