Andrea Rentmeister

Chemo-enzymatic fluorination of unactivated organic compounds

Fluorination has gained an increasingly important role in drug discovery and development. Here we describe a versatile strategy that combines cytochrome P450-catalyzed oxygenation with deoxofluorination to achieve mono- and polyfluorination of nonreactive sites in a variety of organic scaffolds. This procedure was applied for the rapid identification of fluorinated drug derivatives with enhanced membrane permeability.

GGA1 Is Expressed in the Human Brain and Affects the Generation of Amyloid β-Peptide

The β-amyloid peptide (Aβ) is a major component of Alzheimer disease (AD)-associated senile plaques and is generated by sequential cleavage of the β-amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase. BACE1 cleaves APP at the N terminus of the Aβ domain, generating a membrane-bound C-terminal fragment (CTF-β) that can be subsequently cleaved by γ-secretase within the transmembrane domain to release Aβ. Because BACE1 initiates Aβ generation, it represents a potential target molecule to interfere with Aβ production in therapeutic strategies for AD. BACE1 interacts with Golgi-localized, γ-ear-containing, ADP ribosylation factor-binding (GGA) proteins that are involved in the subcellular trafficking of BACE1. Here, we show that GGA1 is preferentially expressed in neurons of the human brain. GGA1 was also detected in activated microglia surrounding amyloid plaques in AD brains. Functional analyses with cultured cells demonstrate that GGA1 is implicated in the proteolytic processing of APP. Overexpression of GGA1 or a dominant-negative variant reduced cleavage of APP by BACE1 as indicated by a decrease in CTF-β generation. Importantly, overexpression of GGA1 reduced, whereas RNAi-mediated suppression of GGA1 increased the secretion of Aβ. The modulation of APP processing by GGA1 is independent of a direct interaction of both proteins. Because total cellular activity of BACE1 was not affected by GGA1 expression, our data indicate that changes in the subcellular trafficking of BACE1 or other GGA1-dependent proteins contribute to changes in APP processing and Aβ generation. Thus, GGA proteins might be involved in the pathogenesis of AD.

Cell-specific aptamers as emerging therapeutics.

Aptamers are short nucleic acids that bind to defined targets with high affinity and specificity. The first aptamers have been selected about two decades ago by an in vitro process named SELEX (systematic evolution of ligands by exponential enrichment). Since then, numerous aptamers with specificities for a variety of targets from small molecules to proteins or even whole cells have been selected. Their applications range from biosensing and diagnostics to therapy and target-oriented drug delivery. More recently, selections using complex targets such as live cells have become feasible. This paper summarizes progress in cell-SELEX techniques and highlights recent developments, particularly in the field of medically relevant aptamers with a focus on therapeutic and drug-delivery applications.

Conformational changes in the expression domain of the Escherichia coli thiM riboswitch

The thiM riboswitch contains an aptamer domain that adaptively binds the coenzyme thiamine pyrophosphate (TPP). The binding of TPP to the aptamer domain induces structural rearrangements that are relayed to a second domain, the so-called expression domain, thereby interfering with gene expression. The recently solved crystal structures of the aptamer domains of the thiM riboswitches in complex with TPP revealed how TPP stabilizes secondary and tertiary structures in the RNA ligand complex. To understand the global modes of reorganization between the two domains upon metabolite binding the structure of the entire riboswitch in presence and absence of TPP needs to be determined. Here we report the secondary structure of the entire thiM riboswitch from Escherichia coli in its TPP-free form and its transition into the TPP-bound variant, thereby depicting domains of the riboswitch that serve as communication links between the aptamer and the expression domain. Furthermore, structural probing provides an explanation for the lack of genetic control exerted by a riboswitch variant with mutations in the expression domain that still binds TPP.

An Aptamer Targeting the Apical-Loop Domain Modulates pri-miRNA Processing†

MicroRNAs (miRNAs) are short noncoding RNAs that recognize complementary bases on target mRNAs, thereby triggering either inhibition of translation initiation or mRNA degradation. They have unique expression patterns and are involved in almost every important biological process, including cell proliferation, differentiation, and apoptosis. In turn, deregulation of miRNA expression patterns is a key condition in the onset and progression of tumor development. Following the synthesis of the primary transcript (primiRNA), the maturation process of miRNAs comprises several steps. First, the pri-miRNA is hydrolyzed by the microprocessor complex, consisting of Drosha/DGCR8, to release hairpin-shaped precursor RNAs (pre-miRNAs). Subsequently, the pre-miRNA is exported into the cytoplasm and further processed by the type III ribonuclease Dicer to produce mature miRNAs. Owing to the prominent role of miRNAs in regulating gene expression, considerable efforts have been made to develop selective tools that will allow the direct targeting of miRNAs affecting either their biogenesis or function. One such class of tools is represented by the so-called antagomirs, short single-stranded 2’-methoxy-modified oligonucleotides. Antagomirs recognize mature miRNAs by complementary bases, thereby preventing miRNA–mRNA association. Here, we introduce another class of nucleic acid based molecular tools to interfere with miRNA activity, namely, RNA aptamers that specifically recognize the loop domains of a pri-miRNA and modulate its processing. Aptamers are short single-stranded nucleic acids that fold into well-defined three-dimensional structures that facilitate specific target recognition. Aptamers can be isolated by an in vitro selection process, and a wide variety of target molecules, such as proteins, cells, small molecules, and nucleic acids have been already applied for aptamer identification. Especially in the latter case, the interaction between the aptamer and the target RNA has been proven to rely on fitting three-dimensional shapes, going beyond mere recognition through complementary base pairing. We sought to elucidate whether RNA aptamers could be used as an alternative nucleic acid based molecular tool to specifically interfere with the biogenesis of individual miRNAs. Here we describe the isolation and characterization of an RNA aptamer that specifically targets the pri-miRNA polycistron 17~ 18a~ 19a~ 20a~ 19b-1~ 92. We show that the aptamer binds inter alia to the apical-loop domain of primiR18a and thereby inhibits the biogenesis of all miRNAs 1719b-1 within this cluster. Our results show that aptamers can be applied as agents that modulate pri-miRNA processing and as tools for elucidating mechanisms of this process. Furthermore, the ability to modulate the maturation of miRNA by targeting the apical-loop domain supports the importance of these domains during pri-miRNA processing. To obtain aptamers that specifically target pri-miR17~ 18a~ 19a~ 20a~ 19b-1~ 92, we applied an in vitro selection scheme in which the 791 nucleotide (nt) miRNA polycistron comprising pri-miR17 ~ 18a ~ 19a~ 20a ~ 19b-1 was biotinylated at its 5’-end and immobilized on streptavidin-coated magnetic beads (Figure 1). The beads were incubated with an RNA library comprising a 25nt random region. To avoid the participation of the constant regions of the RNA library in pri-miRNA binding, we sequestered these regions by hybridization to complementary oligodeoxynucleotides (ODNs), leaving the random nucleotides free for independent folding. After removal of all unbound RNA sequences, the retained RNAs were eluted by adding EDTA. This step essentially favors the release of those molecules that require Mg ions for RNA binding over those that exclusively rely on complementary base pairing. After seven selection cycles, enhanced pri-miRNA binding was detectable, which could be further improved by five succeeding cycles of selection and amplification (Figure S1A in the Supporting Information). The RNA library obtained from selection cycle 12 was cloned and sequenced. Amongst 17 analyzed sequences, nine revealed the consensus motif I, 5’-AACACCUC, comple[*] C. E. L nse, C. S. Hopp, Dr. A. Rentmeister, Prof. M. Famulok, Prof. G. Mayer Life and Medical Sciences (LIMES), University of Bonn Gerhard-Domagk-Strasse 1, Bonn (Germany) Fax: (+49)228-734-809 E-mail: m.famulok@uni-bonn.de gmayer@uni-bonn.de

Genetically encoded tools for RNA imaging in living cells.

RNA imaging probes help us investigate how transport and dynamics of RNA contribute to subcellular RNA localization or regulation of gene expression. Out of the plethora of strategies that have been developed to image RNA in living cells, genetically encoded probes are interesting because they can be produced by the cellular machinery and do not require transfection of the cell. These probes can be grouped into fluorophore-binding aptamers and RNA-binding proteins fused to whole or split fluorescent proteins. In this review, we highlight recent developments in the field of genetically encoded probes for RNA imaging and discuss the strengths and limitations of the different approaches.

Current Approaches for RNA Labeling in Vitro and in Cells Based on Click Reactions

Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments.

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination.

Sequence diversity within a family of functional enzymes provides a platform for elucidating structure–function relationships and for protein engineering to improve properties important for applications. Access to natures vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) – Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS – to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence–property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure‐guided recombination can play in helping to identify sequence–function relationships within a family of enzymes by supplementing natural diversity with synthetic diversity.

Engineered Bacterial Mimics of Human Drug Metabolizing Enzyme CYP2C9

Simple and universal methods for the preparation of human drug metabolites are required to produce quantities sufficient for their characterization and toxicity testing. Synthetic chemistry lacks general catalysts for selective oxidation of unactivated CH bonds, a transformation that plays a key role in metabolism; bioconversions using P450 enzymes have emerged as a powerful alternative. Variants of P450BM3 from Bacillus megaterium act on diverse substrates, including drugs. Acidic substrates, such as the compounds metabolized by CYP2C9, which is one of three main hepatic human P450s, are not accepted by P450BM3 variants engineered to date. Herein, we report bacterial mimics of CYP2C9, which are active on two widely administered drugs, naproxen and ibuprofen, that are CYP2C9 substrates in vivo. These P450BM3 variants can also act on desmethylnaproxen, the human metabolite of naproxen, and convert it to the 1,4‐naphthoquinone derivative. We analyzed the crystal structure of the heme domain of an early intermediate in the directed‐evolution experiment. The active site mutation, L75R, which initially conferred activity on charged substrates, dramatically increased structural flexibility in the B′‐helix. This increased flexibility, which was accompanied by a dramatic decrease in enzyme stability, may contribute to the variant’s ability to accept a broader range of substrates.

Current covalent modification methods for detecting RNA in fixed and living cells.

Labeling RNAs is of particular interest for elucidating localization, transport, and regulation of specific transcripts, ideally in living cells. Numerous methods have been developed ranging from hybridizing probes to genetically encoded reporters and chemo-enzymatic approaches. This review focuses on covalent labeling approaches that rely on the introduction of a small reactive group into the nascent or completed transcript followed by bioorthogonal click chemistry. State of the approaches for labeling RNA in fixed and living cells will be presented and emerging strategies with great potential for application in the complex cellular environment will be discussed.