Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Ribonucleoprotein purification and characterization using RNA Mango.

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.

Structural basis for high-affinity fluorophore binding and activation by RNA Mango.

Genetically encoded fluorescent protein tags have revolutionized proteome studies, whereas the lack of intrinsically fluorescent RNAs has hindered transcriptome exploration. Among several RNA-fluorophore complexes that potentially address this problem, RNA Mango has an exceptionally high affinity for its thiazole orange (TO)-derived fluorophore, TO1-Biotin (Kd ∼3 nM), and, in complex with related ligands, it is one of the most redshifted fluorescent macromolecular tags known. To elucidate how this small aptamer exhibits such properties, which make it well suited for studying low-copy cellular RNAs, we determined its 1.7-Å-resolution co-crystal structure. Unexpectedly, the entire ligand, including TO, biotin and the linker connecting them, abuts one of the near-planar faces of the three-tiered G-quadruplex. The two heterocycles of TO are held in place by two loop adenines and form a 45° angle with respect to each other. Minimizing this angle would increase quantum yield and further improve this tool for in vivo RNA visualization.

RNA complex purification using high-affinity fluorescent RNA aptamer tags.

RNA plays important roles in cellular processes, but RNA-protein complexes are notoriously hard to isolate and study. We compare and contrast existing RNA- and protein-purification strategies with the potential of new RNA-tagging systems such as RNA Spinach and RNA Mango. Each RNA aptamer binds a small fluorophore, resulting in a highly fluorescent complex that is thousands of times brighter than the unbound fluorophore. Provided that the aptamer binding affinity is high enough, derivatized dyes can be used in conjunction with these aptamers to purify RNA complexes while simultaneously using their intrinsic fluorescence to track the complex of interest. The known strengths and weakness of these RNA tagging systems are discussed.

RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes.

E. coli 6S RNA release from RNA polymerase requires σ70 ejection by scrunching and is orchestrated by a conserved RNA hairpin.

The 6S RNA in Escherichia coli suppresses housekeeping transcription by binding to RNA polymerase holoenzyme (core polymerase + σ7°) under low nutrient conditions and rescues σ7°-dependent transcription in high nutrient conditions by the synthesis of a short product RNA (pRNA) using itself as a template. Here we characterize a kinetic intermediate that arises during 6S RNA release. This state, consisting of 6S RNA and core polymerase, is related to the formation of a top-strand "release" hairpin that is conserved across the γ-proteobacteria. Deliberately slowing the intrinsic 6S RNA release rate by nucleotide feeding experiments reveals that σ7° ejection occurs abruptly once a pRNA length of 9 nucleotides (nt) is reached. After σ7° ejection, an additional 4 nt of pRNA synthesis is required before the 6S:pRNA complex is finally released from core polymerase. Changing the E. coli 6S RNA sequence to preclude formation of the release hairpin dramatically slows the speed of 6S RNA release but, surprisingly, does not alter the abruptness of σ7° ejection. Rather, the pRNA size required to trigger σ7° release increases from 9 nt to 14 nt. That a precise pRNA length is required to trigger σ7° release either with or without a hairpin implicates an intrinsic "scrunching"-type release mechanism. We speculate that the release hairpin serves two primary functions in the γ-proteobacteria: First, its formation strips single-stranded "-10" 6S RNA interactions away from σ7°. Second, the formation of the hairpin accumulates RNA into a region of the polymerase complex previously associated with DNA scrunching, further destabilizing the 6S:pRNA:polymerase complex.