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Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

ABSTRACT: Two classes of riboswitches related to the ykkC guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the mutant, the base of ppGpp replaces G96 in three-dimensional space. This disrupts the S-turn, which is a primary structural feature of the ykkC RNA motif. These dramatic differences in ligand specificity are achieved with minimal mutations. ykkC aptamers are therefore a prime example of an RNA fold with a rugged fitness landscape. The ease with which the ykkC aptamer acquires new specificity represents a striking case of evolvability in RNA.

SUBMITTER: Knappenberger AJ

PROVIDER: S-EPMC6031431 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

Knappenberger Andrew John AJ Reiss Caroline Wetherington CW Strobel Scott A SA

eLife 20180607

Two classes of riboswitches related to the <i>ykkC</i> guanidine-I riboswitch bind phosphoribosyl pyrophosphate (PRPP) and guanosine tetraphosphate (ppGpp). Here we report the co-crystal structure of the PRPP aptamer and its ligand. We also report the structure of the G96A point mutant that prefers ppGpp over PRPP with a dramatic 40,000-fold switch in specificity. The ends of the aptamer form a helix that is not present in the guanidine aptamer and is involved in the expression platform. In the ...[more]

PMID: 29877798

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Project description:In this study, two respective groups of RNA aptamers have been selected against two main classes of glycosaminoglycans (GAGs), heparosan, and chondroitin, as they have proven difficult to specifically detect in biological samples. GAGs are linear, anionic, polydisperse polysaccharides found ubiquitously in nature, yet their detection remains problematic. GAGs comprised repeating disaccharide units, consisting of uronic acid and hexosamine residues that are often also sulfated at various positions. Monoclonal antibodies are frequently used in biology and medicine to recognize various biological analytes with high affinity and specificity. However, GAGs are conserved across the whole animal phylogenic tree and are nonimmunogenic in hosts traditionally used for natural antibody generation. Thus, it has been challenging to obtain high affinity, selective antibodies that recognize various GAGs. In the absence of anti-GAG antibodies, glycobiologists have relied on the use of specific enzymes to convert GAGs to oligosaccharides for analysis by mass spectrometry. Unfortunately, while these methods are sensitive, they can be labor-intensive and cannot be used for in situ detection of intact GAGs in cells and tissues. Aptamers are single-stranded oligonucleotide (DNA or RNA) ligands capable of high selectivity and high affinity detection of biological analytes. Aptamers can be developed in vitro by the systematic evolution of ligands by exponential enrichment (SELEX) to recognize nonimmunogenic targets, including neutral carbohydrates. This study utilizes the SELEX method to generate RNA aptamers, which specifically bind to the unmodified GAGs, heparosan, and chondroitin. Binding confirmation and cross-screening with other GAGs were performed using confocal microscopy to afford three specific GAGs to each target. Affinity constant of each RNA aptamer was obtained by fluorescent output after interaction with the respective GAG target immobilized on plates; the K D values were determined to be 0.71-1.0 μM for all aptamers. Upon the success of chemical modification (to stabilize RNA aptamers in actual biological systems) and fluorescent tagging (to only visualize RNA aptamers) of these aptamers, they would be able to serve as a specific detection reagent of these important GAGs in biological samples.

Project description:BackgroundThe human mouth is a natural laboratory for studying how bacterial communities differ across habitats. Different bacteria colonize different surfaces in the mouth-teeth, tongue dorsum, and keratinized and non-keratinized epithelia-despite the short physical distance between these habitats and their connection through saliva. We sought to determine whether more tightly defined microhabitats might have more tightly defined sets of resident bacteria. A microhabitat may be characterized, for example, as the space adjacent to a particular species of bacterium. Corncob structures of dental plaque, consisting of coccoid bacteria bound to filaments of Corynebacterium cells, present an opportunity to analyze the community structure of one such well-defined microhabitat within a complex natural biofilm. Here, we investigate by fluorescence in situ hybridization and spectral imaging the composition of the cocci decorating the filaments.ResultsThe range of taxa observed in corncobs was limited to a small subset of the taxa present in dental plaque. Among four major groups of dental plaque streptococci, two were the major constituents of corncobs, including one that was the most abundant Streptococcus species in corncobs despite being relatively rare in dental plaque overall. Images showed both Streptococcus types in corncobs in all individual donors, suggesting that the taxa have different ecological roles or that mechanisms exist for stabilizing the persistence of functionally redundant taxa in the population. Direct taxon-taxon interactions were observed not only between the Streptococcus cells and the central corncob filament but also between Streptococcus cells and the limited subset of other plaque bacteria detected in the corncobs, indicating species ensembles involving these taxa as well.ConclusionsThe spatial organization we observed in corncobs suggests that each of the microbial participants can interact with multiple, albeit limited, potential partners, a feature that may encourage the long-term stability of the community. Additionally, our results suggest the general principle that a precisely defined microhabitat will be inhabited by a small and well-defined set of microbial taxa. Thus, our results are important for understanding the structure and organizing principles of natural biofilms and lay the groundwork for future work to modulate and control biofilms for human health. Video Abstract.

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Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

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Structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of RNA.

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