Clemens Richert

Universität Stuttgart, Stuttgart, Baden-Württemberg, Germany

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Publications (140)783.27 Total impact

  • Sven Vollmer · Clemens Richert

    No preview · Article · Dec 2015 · Chemistry - A European Journal
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    Sven Vollmer · Clemens Richert
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    ABSTRACT: Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA.
    Preview · Article · Nov 2015 · Chemistry - A European Journal
  • Mario Jauker · Helmut Griesser · Clemens Richert
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    ABSTRACT: Wie sich die heutige biochemische Maschinerie aus einfachen Vorläufern entwickelte, ist eine ungelöste Frage. Wir zeigen nun, dass Ribonukleotide und Aminosäuren in Abwesenheit von Enzymen zu Peptidyl-RNAs kondensieren, und zwar unter Bedingungen, die auch spontanes genetisches Kopieren induzieren. Es treten templatfreie Bildung von RNA-Strängen, die genetische Information codieren können, die Bildung von Peptidyl-RNAs und die Bildung der Cofaktoren NAD+, FAD und ATP unter den gleichen Bedingungen auf. In den Peptidyl-RNAs sind die Peptidketten über eine Phosphorsäureamidgruppe an ein Ribonukleotid gebunden. Peptidyl-RNAs mit langen Peptidketten lassen sich aus einem Pool selektieren, wenn eine lipophile Phase angeboten wird, die das Innere von Membranen simuliert. Freie Peptide lassen sich aus Peptidyl-RNA durch Ansäuern freisetzen. Unsere Ergebnisse zeigen, dass Schlüsselmoleküle der Genetik, der Katalyse und des Metabolismus unter denselben Bedingungen entstehen können, ohne dass es mineralische Oberflächen, Enzyme oder eine chemische Voraktivierung bräuchte.
    No preview · Article · Oct 2015 · Angewandte Chemie
  • Mario Jauker · Helmut Griesser · Clemens Richert

    No preview · Article · Oct 2015
  • Mario Jauker · Helmut Griesser · Clemens Richert
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    ABSTRACT: How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD(+) , FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate-linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre-activation.
    No preview · Article · Oct 2015 · Angewandte Chemie International Edition
  • Mario Jauker · Helmut Griesser · Clemens Richert
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    ABSTRACT: Template-directed incorporation of nucleotides at the terminus of a growing complementary strand is the basis of replication. For RNA, this process can occur in the absence of enzymes, if the ribonucleotides are first converted to an active species with a leaving group. Thus far, the activation required a separate chemical step, complicating prebiotically plausible scenarios. Here we show that a combination of a carbodiimide and an organocatalyst induces near-quantitative incorporation of any of the four ribonucleotides. Upon in situ activation, adenosine monophosphate was found to also form oligomers in aqueous solution. So, both de novo strand formation and sequence-specific copying can occur without an artificial synthetic step.
    No preview · Article · Oct 2015 · Angewandte Chemie International Edition
  • Anja Göckel · Clemens Richert
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    ABSTRACT: Nicotinamide adenine dinucleotide (NAD) is a pivotal redox cofactor of primary metabolism. Its redox reactivity is based on the nicotinamide mononucleotide (NMN) moiety. We investigated whether NMN(+) can engage in pairing interactions, when incorporated into an oligonucleotide. Here we describe the incorporation of NMN(+) at the 3'-terminus of an oligodeoxynucleotide via a phosphoramidate coupling in solution. The stability of duplexes and triplexes with the NMN(+)-containing strand was measured in UV-melting curves. While the melting points of duplexes with different bases facing the nicotinamide were similar, triplex stabilities varied greatly between different base combinations, suggesting specific pairing. The most stable triplexes were found when a guanine and an adenine were facing the NMN(+) residue. Their triplex melting points were higher than those of the corresponding triplexes with a thymidine residue at the same position. These results show that NMN(+) residues can be recognized selectively in DNA helices and are thus compatible with the molecular recognition in nucleic acids.
    No preview · Article · Sep 2015 · Organic & Biomolecular Chemistry
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    ABSTRACT: This chapter focuses on the structure and stabilization of triplex DNA. The canonical DNA triplex structure is generally formed between an oligonucleotide and a homopurine– homopyrimidine duplex. The third strand (triplex-forming oligonucleotide (TFO)) binds in the major groove of the duplex and forms hydrogen bonds with the hydrogen bond donor and acceptor groups available on the major groove edge of the purine bases. According to where the TFO originates from, triplexes can be classified as intramolecular and intermolecular triplexes. The third strand in the triplex can also result from an exogenously applied molecule to form an intermolecular triplex structure. The pH of the solution is an essential parameter for the CGC+ triplet. Basic oligopeptides, another class of biologically relevant polycations, can stabilize triplex DNA. Cationic amino acid residues of basic peptides might bind to and neutralize phosphate groups of triple-helical nucleic acids. One important emerging field is the use of synthetic molecules to tune and modify the stability, functionality, and assembly of DNA-based structures. These molecules can be incorporated into the structures through one of two methods: covalent insertion or non-covalent interactions. Due to the ease of automated synthesis and the efficiency of coupling approaches, a plethora of synthetic modifications are available. Insertion of synthetic linkers has several direct effects on the DNA duplexes. Covalently inserting synthetic molecules into the DNA backbone introduces many unique properties for DNA assembly and represents a powerful tool towards controlling structure. For the purposes of guiding DNA assembly, groove binders and intercalators have interesting properties. The application of these properties to DNA self-assembly is discussed in this chapter. Intercalators and groove binders have demonstrated the ability to stabilize fully duplexed structures, modify assembly outcomes, increase yields, functionalize assemblies, and connect blunt-ended duplexes. Oligodeoxynucleotides are readily synthesized in automatic fashion in lengths of up to 100–50 nucleotides, and they form antiparallel duplex structures, based on the base-paring rules of Watson and Crick. Duplexes of DNA strands have a typical persistence length in aqueous buffer of over 100 base pairs. Shortly after the publication on assemblies of hybrids with dimers as DNA arms, Seeman and coworkers succeeded in crystallizing DNA folding motifs into lattices with designed crystal structure. In order to develop the next generation of hybrids, the authors sought a collaboration with theoreticians. Wolfgang Wenzel and coworkers took it upon themselves to develop a system for simulating the assembly of DNA hybrids, using a coarse-grained model. The strength of the association of the second-generation hybrids warranted a special approach for melting curves. Branched oligonucleotide hybrids with rigid cores will remain interesting only if they show their usefulness in practical applications. Controlled assembly of soft nanoparticles requires a recognition event to trigger the formation of a non-covalent assembly. A large number of soft nanoparticles are known, and among the most important class of natural soft nanoparticles are vesicles. Vesicles are lipid nanoparticles based on natural lipids and are of particular interest due to their occurrence in nature as intraor extracellular transport vehicles. However, soft nanoparticles based on lipid bilayers possess a universal mode for encoding surfaces by membrane anchoring of lipid modified DNA. In light of the technically demanding procedures for solid nanoparticles, non-covalent attachment of DNA to lipid bilayer surfaces becomes a very attractive technology. Incorporation of one or more lipid-membrane anchors into DNA leads to different sequence designs, which in general all allow assembly of liposomes. Ultraviolet spectroscopy in the presence of liposomes allows monitoring of DNA controlled assembly processes based on double or triple helix formation. This chapter provides a concise overview of the delicate relationship between biologically relevant metal ions or metal complexes and the two classes of naturally occurring functional RNAs: ribozymes or riboswitches. It first introduces the most important types of metal ion– RNA interactions, concentrating on the biologically most significant metal ions and discussing the various coordination modes and most frequent specific binding sites. The chapter then presents the world of both small and large ribozymes giving special attention to the influence of cations on the structure, folding, and function. It further explains how metal ions and metal complexes are involved in the correct structure formation and functioning of riboswitches. Riboswitches usually bind only a single metabolite to undergo the structural rearrangement required for gene regulation. The mgtA and the M-box riboswitches are well-defined examples of metal-sensing RNA. A major research area in DNA nanotechnology involves the development of DNA switching systems and DNA machines. This chapter exemplifies several principles to assemble DNA switching devices and DNA machines and discusses the potential applications. It highlights the existing structural and functional “tool-boxes” of nucleic acids to assemble DNA switches and machines. The chapter explains the perspectives of the area within the broad topic of DNA nanotechnology. It exemplifies the tailoring of DNA switches by ions or physical triggers such as photonic or electrical signals. The use of DNA switches and machines in nano-medicine has sparked substantial interest and several preliminary reports highlighting the future potential of such systems. The dynamic and switchable control of the organization of plasmonic particles or fluorophore–plasmonicnanoparticle conjugates holds great promise in material science. DNA switches and machines are expected to provide effective scaffolds for programmed synthesis by the dictated stimuli-triggered interactions of chemical reactants. This chapter describes the use of DNA as chiral bio-scaffold in the design of hybrid catalysts and their application in asymmetric catalysis. Some current and relevant examples are discussed, followed by an overview of mechanistic studies. DNA-based asymmetric catalysis is an exponent of the general concept of hybrid catalysts, which aims to merge the attractive properties of homogeneous and bio-catalysis. There are two main approaches to the anchoring of a transition metal complex to DNA. In covalent anchoring, the ligand for the metal is attached to the DNA via a chemical bond. A particularly attractive aspect of supramolecular anchoring is the easy formation of the catalyst since it involves spontaneous self-assembly of the transition metal complex with DNA. Moreover, in this approach it is usually DNA from natural sources, such as calf thymus or salmon testes DNA, that is used.
    No preview · Article · Jul 2015
  • Alexander Schwenger · Wolfgang Frey · Clemens Richert
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    ABSTRACT: Molecular storage solutions for incorporating small molecules in crystalline matrices are of interest in the context of structure elucidation, decontamination, and slow release of active ingredients. Here we report the syntheses of 1,3,5,7-tetrakis(2,4-dimethoxyphenyl)adamantane, 1,3,5,7-tetrakis(4-methoxyphenyl)adamantane, 1,3,5,7-tetrakis(4-methoxy-2-methylphenyl)adamantane, and 1,3,5,7-tetrakis(4-methoxy-2-ethylphenyl)adamantane, together with their X-ray crystal structures. All four compounds crystallize readily. Only the octaether shows an unusual level of (pseudo)polymorphism in its crystalline state, combined with the ability to include a number of different small molecules in its crystal lattices. A total of 20 different inclusion complexes with guest molecules as different as ethanol or trifluorobenzene were found. For nitromethane and benzene, schemes for uptake and release are presented. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    No preview · Article · Apr 2015 · Chemistry - A European Journal
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    ABSTRACT: Oligonucleotide hybrids with organic cores as rigid branching elements and four or six CG dimer strands have been shown to form porous materials from dilute aqueous solution. In order to explore the limits of this form of DNA-driven assembly, we prepared hybrids with three or eight DNA arms via solution-phase syntheses, using H-phosphonates of protected dinucleoside phosphates. This included the synthesis of (CG)8TREA, where TREA stands for the tetrakis[4-(resorcin-5-ylethynyl)phenyl]adamantane core. The ability of the new compounds to assemble in a DNA-driven fashion was studied by UV-melting analysis and NMR, using hybrids with self-complementary CG zipper arms or non-self-complementary TC dimer arms. The three-arm hybrid failed to form a material under conditions where four-arm hybrids did so. Further, the assembly of TREA hybrids appears to be dominated by hydrophobic interactions, not base pairing of the DNA arms. These results help in the design of materials forming by multivalent DNA-DNA interactions.
    No preview · Article · Dec 2014 · The Journal of Organic Chemistry
  • Claudia Gerlach · Birgit Claasen · Clemens Richert
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    ABSTRACT: Binding RNA targets, such as microRNAs, with high fidelity is challenging, particularly when the nucleobases to be bound are located at the terminus of the duplex between probe and target. Recently, a peptidyl chain terminating in a quinolone, called ogOA, was shown to act as a cap that enhances affinity and fidelity for RNAs, stabilizing duplexes with Watson-Crick pairing at their termini. Here we report the three-dimensional structure of an intramolecular complex between a DNA strand featuring the ogOA cap and an RNA segment, solved by NMR and restrained torsion angle molecular dynamics. The quinolone stacks on the terminal base pair of the hybrid duplex, positioned by the peptidyl chain, whose prolinol residue induces a sharp bend between the 5' terminus of the DNA chain and the glycine linked to the oxolinic acid residue. The structure explains why canonical base pairing is favored over hard-to-suppress mismatched base combinations, such as T:G and A:A, and helps to design improved high-fidelity probes for RNA.
    No preview · Article · Nov 2014 · ChemBioChem
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    ABSTRACT: It is becoming increasingly clear that nature uses RNAs extensively for regulating vital functions of the cell, and short sequences are frequently used to suppress gene expression. However, controlling the concentration of small molecules intracellularly through designed RNA sequences that fold into ligand-binding structures is difficult. The development of “endless”, a triplex-based folding motif that can be expressed in mammalian cells and binds the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP), is described. In vitro, DNA or RNA versions of endless show low micromolar to nanomolar dissociation constants for cGMP. To test its functionality in vivo, four endless RNA motifs arranged in tandem were co-expressed with a fluorescent cGMP sensor protein in murine vascular smooth muscle cells. Nitric oxide induced endogenous cGMP signals were suppressed in endless-expressing cells compared to cells expressing a control motif, which suggests that endless can act as a genetically encoded cGMP sink to modulate signal transduction in cells.
    Full-text · Article · Aug 2014 · Angewandte Chemie International Edition
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    ABSTRACT: It is becoming increasingly clear that nature uses RNAs extensively for regulating vital functions of the cell, and short sequences are frequently used to suppress gene expression. However, controlling the concentration of small molecules intracellularly through designed RNA sequences that fold into ligand-binding structures is difficult. The development of “endless”, a triplex-based folding motif that can be expressed in mammalian cells and binds the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP), is described. In vitro, DNA or RNA versions of endless show low micromolar to nanomolar dissociation constants for cGMP. To test its functionality in vivo, four endless RNA motifs arranged in tandem were co-expressed with a fluorescent cGMP sensor protein in murine vascular smooth muscle cells. Nitric oxide induced endogenous cGMP signals were suppressed in endless-expressing cells compared to cells expressing a control motif, which suggests that endless can act as a genetically encoded cGMP sink to modulate signal transduction in cells.
    No preview · Article · Aug 2014 · Angewandte Chemie
  • Marco Minuth · Wolfgang Frey · Clemens Richert
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    ABSTRACT: Salicylic aldehyde C-nucleosides, when incorporated in oligodeoxynucleotides, have been widely used to prepare DNA duplexes with metallo base pairs. Here, we report a synthesis of such nucleosides that involves diastereoselective formation of the glycosidic bond via Heck reaction.
    No preview · Article · Jun 2014 · Synlett
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    Eric Kervio · Birgit Claasen · Ulrich E Steiner · Clemens Richert
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    ABSTRACT: The transmission of genetic information relies on Watson–Crick base pairing between nucleoside phosphates and template bases in template–primer complexes. Enzyme-free primer extension is the purest form of the transmission process, without any chaperon-like effect of polymerases. This simple form of copying of sequences is intimately linked to the origin of life and provides new opportunities for reading genetic information. Here, we report the dissociation constants for complexes between (deoxy)nucleotides and template–primer complexes, as determined by nuclear magnetic resonance and the inhibitory effect of unactivated nucleotides on enzyme-free primer extension. Depending on the sequence context, Kd′s range from 280 mM for thymidine monophosphate binding to a terminal adenine of a hairpin to 2 mM for a deoxyguanosine monophosphate binding in the interior of a sequence with a neighboring strand. Combined with rate constants for the chemical step of extension and hydrolytic inactivation, our quantitative theory explains why some enzyme-free copying reactions are incomplete while others are not. For example, for GMP binding to ribonucleic acid, inhibition is a significant factor in low-yielding reactions, whereas for amino-terminal DNA hydrolysis of monomers is critical. Our results thus provide a quantitative basis for enzyme-free copying.
    Full-text · Article · May 2014 · Nucleic Acids Research
  • Christoph Kröner · Anja Göckel · Wenjing Liu · Clemens Richert
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    ABSTRACT: Glowing when reaching body temperature The surface of a sepharose bead with immobilized triplex motifs releases bound NADH when approaching 37 °C. The NADH then induces bioluminescence. For more details, see the Full Paper by C. Richert et al. on page 15879 ff.
    No preview · Article · Nov 2013 · Chemistry
  • Christoph Kröner · Anja Göckel · Wenjing Liu · Clemens Richert
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    ABSTRACT: Cofactors are pivotal compounds for the cell and many biotechnological processes. It is therefore interesting to ask how well cofactors can be bound by oligonucleotides designed not to convert but to store and release these biomolecules. Here we show that triplex-based DNA binding motifs can be used to bind nucleotides and cofactors, including NADH, FAD, SAM, acetyl CoA, and tetrahydrofolate (THF). Dissociation constants between 0.1 μM for SAM and 35 μM for THF were measured. A two-nucleotide gap still binds NADH. The selectivity for one ligand over the others can be changed by changing the sequence of the binding pocket. For example, a mismatch placed in one of the two triplets adjacent to the base-pairing site changes the selectivity, favoring the binding of FAD over that of ATP. Further, changing one of the two thymines of an A-binding motif to cytosine gives significant affinity for G, whereas changing the other does not. Immobilization of DNA motifs gives beads that store NADH. Exploratory experiments show that the beads release the cofactor upon warming to body temperature.
    No preview · Article · Nov 2013 · Chemistry - A European Journal
  • Rüdiger Haug · Markus Kramer · Clemens Richert
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    ABSTRACT: TPP oligonucleotides: Hybridization probes that interrogate target sequences through base pairing, stacking on the terminus, and binding in the minor groove are presented. All subunits of the probes contribute to the target affinity, leading to melting point increases of up to 45 °C. (TPP=three-pronged probe).
    No preview · Article · Nov 2013 · Chemistry - A European Journal
  • Marco Minuth · Clemens Richert
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    ABSTRACT: Shaping up for an A: Adenine is the only canonical nucleobase that does not offer a third hydrogen-bonding functionality at its Watson-Crick face, making it difficult to bind with high affinity. A 6-ethynyl-2-pyridone binds more tightly and with greater sequence fidelity than thymine. VdW=van der Waals interactions.
    No preview · Article · Oct 2013 · Angewandte Chemie International Edition
  • Heike Vogel · Claudia Gerlach · Clemens Richert
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    ABSTRACT: Rapid, template-directed ligation reactions between a phosphate-terminated oligonucleotide and an unphosphorylated reaction partner may be induced by cyanogen bromide (BrCN). Frequently, however, the reaction is low yielding, and even a large excess of the condensing agent can fail to induce quantitative conversions. In this study, we used BrCN to induce chemical primer extension reactions. Here, we report that buffers containing hydroxyl groups react with short oligodeoxynucleotides in the presence of BrCN. One stable adduct between HEPBS buffer and cytosine was characterized by mass spectrometry and NMR after HPLC purification, indicating that a side reaction occurred at this nucleobase. Further, a first example of a primer extension reaction between an unmodified oligodeoxynucleotide as primer and dGMP is reported. Together, our results shed light on the potency, as well as the drawbacks of BrCN as a highly reactive condensing reagent for the ligation of unmodified nucleic acids.
    No preview · Article · Jan 2013 · Nucleosides Nucleotides & Nucleic Acids

Publication Stats

2k Citations
783.27 Total Impact Points

Institutions

  • 2008-2015
    • Universität Stuttgart
      • Institute of Organic Chemistry
      Stuttgart, Baden-Württemberg, Germany
  • 2000-2010
    • Universität Konstanz
      • Department of Chemistry
      Constance, Baden-Württemberg, Germany
  • 1996-2010
    • Tufts University
      • Department of Chemistry
      Georgia, United States
    • Technische Universität München
      München, Bavaria, Germany
  • 2003-2009
    • Karlsruhe Institute of Technology
      • Institute of Organic Chemistry
      Carlsruhe, Baden-Württemberg, Germany
  • 1996-1997
    • Ludwig-Maximilians-University of Munich
      München, Bavaria, Germany
  • 1995-1996
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland
  • 1994
    • University of Cologne
      • Institute of Organic Chemistry
      Köln, North Rhine-Westphalia, Germany