RNA Ligands That Distinguish Metabolite‐Induced Conformations in the TPP Riboswitch

Abstract

Riboswitches are conserved noncoding elements, located in untranslated regions of messenger RNAs (mRNAs), which regulate the expression of metabolism genes in biosynthetic pathways in response to metabolite binding. [1,2] They bind to small metabolite molecules, thereby triggering the expression of distinct genes that are in turn involved in biosynthetic pathways of the metabolite. As riboswitches regulate the expression of essential genes in many bacteria, these RNAs might serve as targets for the development of novel antimicrobial drugs. [3][4][5] The thiM riboswitch of E. coli regulates the expression of the metabolic protein hydroxyethylthiazolkinase and is known to inhibit gene expression mainly by the sequestration of the Shine-Dalgarno (SD) sequence induced by specific binding of thiamine pyrophosphate (TPP) to an aptamer domain.[6] However, like other metabolite-dependent riboswitches, thiM riboswitches also contain an expression domain which does not directly interact with TPP but is required for proper riboswitch function by a mechanism in which TPPinduced structural rearrangements are thought to be relayed to this domain, thereby interfering with gene expression.Crystallographic and NMR spectroscopy analyses of the aptamer domain of TPP riboswitches in complex with its ligand TPP provided insight into how riboswitch aptamer domains create specific binding pockets. [7][8][9] However, to understand how these RNAs function, analysis of the complete TPP riboswitch with and without its ligand is required. However, the ligand-free structure is difficult to refine by NMR spectroscopy or crystallography.[10] Therefore, molecular probes that can distinguish between activation states of riboswitches might help to identify sites that are involved in conformational changes and thus, might be useful for gaining insight into the mechanisms of riboswitch function.One class of molecular probes that can be obtained by in vitro selection are RNA aptamers-single stranded nucleic acids that fold into a distinct 3D-structure and are able to bind with high affinity and specificity to a cognate target molecule.[11] Aptamers have been isolated against a variety of targets including small molecules, peptides, proteins, and living cells [12] and they can discriminate between activation states of proteins.[13] However, no RNA aptamer has been reported to date that is able to differentiate between RNA conformations.Herein, we describe the isolation of short RNA molecules that are able to bind to the TPP-free thiM riboswitch of E. coli and are released upon riboswitch-metabolite complex formation. We applied a modified in vitro selection procedure in which the 165-nucleotide (nt) thiM riboswitch was biotinylated at its 5'-end and immobilized on streptavidin magnetic beads (Scheme 1). These beads were incubated with a RNA library comprising a 25-nt random region. To avoid the participation of the constant regions of the RNA library in thiM binding, we sequestered these regions by hybridization to complementary oligodeoxynucleotide...

Die chemische Biologie von Aptameren

“…Ein weiterer Beweis für die Vielfältigkeit von Nucleinsäuren gelang durch die Entdeckung von kleinen, nichtkodierenden RNA-Molekülen und RNA-Schaltern, die Schlüsselrollen als Regulationselemente bei der Genexpression in Bakterien, Eukaryoten und höheren Organismen spielen. [21][22][23] Besonders RNA-Schalter zeigen, dass die Natur schon seit langem Aptamerdomänen nutzt, um eine hoch affine und spezifische Bindung kleiner Metaboliten sicherzustellen. [24][25][26] Nucleinsäuresequenzen haben eine definierte Funktion, die sich aus ihrer dreidimensionalen Faltung ergibt, und enthalten den Bauplan für ihre Synthese in ihrer eigenen Primärsequenz.…”

“…[23,[43][44][45] [47] Diese Modifizierungen wurden entweder am Phosphat/Ribose-Rückgrat oder an der Nucleobase eingeführt. [48] Der Austausch des DNA-Phosphatrückgrats gegen ein Phosphorthioat erhöht die Stabilität gegen Nucleasen und ermöglicht dadurch den Einsatz solcher Moleküle innerhalb der Zelle.…”

Theoretical studies on the interaction of modified pyrimidines and purines with purine riboswitch

“…It can specifically bind small molecular metabolites to regulate gene expression in virtue of its conformational changes without the need of protein cofactors [1]. Breaker and co-workers [2] firstly discovered the natural riboswitch and demonstrated that some messenger RNAs contain natural aptamer domain.…”

Modulation der pri‐miRNA‐Reifung durch ein die apikale Schleife bindendes Aptamer

“…Vor allem für Nukleinsäuren konnte bewiesen werden, dass sich die Aptamer-Ziel-RNA-Wechselwirkung auf aneinander passende dreidimensionale Formen stützt und nicht nur aufgrund bloßer Erkennung von komplementären Basen zustande kommt. [10][11][12][13] Wir haben untersucht, ob RNA-Aptamere zur spezifischen Beeinflussung der Biogenese einzelner miRNAs genutzt werden können, und beschreiben hier die Isolierung und Charakterisierung eines RNA-Aptamers, das spezifisch an das pri-miRNA-Polycistron 17~18a~19a~20a~19b-19 2 bindet. Wir zeigen, dass das Aptamer unter anderem mit der apikalen Schleife der pri-miR18a wechselwirkt und dadurch die Biogenese aller miRNAs 17-19b-1 innerhalb des Clusters verhindert.…”