Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.
Several turn-on RNA aptamers that activate small molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans-Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to
Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.
There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogenrelated RNA, it requires expensive thermocycling equipment and is comparatively slow. Isothermal methods promise procedural simplicity but have traditionally suffered from amplification artifacts that tend to preclude easy identification of target nucleic acids. Recently, the isothermal SHERLOCK system overcame this problem by using CRISPR to distinguish amplified target sequences from artifactual background signal. However, this system comes at the cost of introducing considerable enzymatic complexity and a corresponding increase in total assay time. Therefore, simpler and less expensive strategies are highly desirable. Here, we demonstrate that by nesting NASBA primers and modifying the NASBA inner primers to encode an RNA Mango aptamer sequence we can dramatically increase the sensitivity of NASBA to 1.5 RNA molecules per microliter. As this isothermal nucleic acid detection scheme directly produces a fluorescent reporter, real-time detection is intrinsic to the assay. Nested Mango NASBA is highly specific and, in contrast to existing RNA detection systems, offers a cheap, simple, and specific way to rapidly detect single-molecule amounts of pathogenic RNA.
splitting of a membrane compartment, is a central theme in biology that manifests during cell division, organelle biogenesis and vesicular transport. Fission involves the local application of forces to bend and constrict or thin down a membrane tube. Since bending requires the bilayer to deviate from its preferred planar configuration, fission is energetically unfavorable. Using reconstitution approaches that involve biochemical screens, we have discovered novel proteins that catalyze fission and have elucidated their mechanism and cellular functions. My talk will describe these recent developments.
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