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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.

Dataset Information

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

Publications

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

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