A class above: In a sophisticated approach to the systematic evolution of ligands by exponential enrichment (SELEX) of cell‐type‐specific nucleic acid aptamers, the title technique was combined with the use of combinatorial nucleic acid libraries to target a defined subpopulation within composite mixtures of cells (see picture). The ssDNA library after 10 selection cycles bound vital Burkitt lymphoma cells more effectively than the starting library.

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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...

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...

No abstract