Clemens Richert

Universität Stuttgart, Stuttgart, Baden-Württemberg, Germany

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Publications (118)604.6 Total impact

  • [Show abstract] [Hide abstract]
    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.
    Angewandte Chemie International Edition 07/2014; · 11.34 Impact Factor
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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.
    Angewandte Chemie 07/2014;
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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.
    Nucleic Acids Research 05/2014; · 8.28 Impact Factor
  • 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).
    Chemistry 11/2013; 19(47):15822-6. · 5.93 Impact Factor
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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.
    Chemistry 11/2013; · 5.93 Impact Factor
  • 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.
    Angewandte Chemie International Edition 08/2013; · 11.34 Impact Factor
  • Andreas Kaiser, Clemens Richert
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    ABSTRACT: Chemical primer extension is the enzyme-free incorporation of nucleotides at the end of an oligonucleotide, directed by a template. The reaction mimics the copying of sequences during replication but relies on recognition and reactivity of nucleic acids alone. Copying is low-yielding, particularly for long RNA. Hydrolysis of active esters and inhibition through hydrolysis products have been identified as factors that prevent high yields, and approaches to overcoming them have culminated in successful template-directed solid-phase syntheses for RNA and phosphoramidate DNA.
    The Journal of Organic Chemistry 01/2013; · 4.56 Impact Factor
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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.
    Chemistry 01/2013; 19(47). · 5.83 Impact Factor
  • 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.
    Nucleosides Nucleotides &amp Nucleic Acids 01/2013; 32(1):17-27. · 0.71 Impact Factor
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    ABSTRACT: Nonenzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5'-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5'-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time), and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. On the basis of these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to 2 orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large "leaps" through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.
    Journal of the American Chemical Society 12/2012; · 10.68 Impact Factor
  • Simone Egetenmeyer, Clemens Richert
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    ABSTRACT: Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5'-O-terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of L-prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step-wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap-bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis. Curr. Protoc. Nucleic Acid Chem. 51:4.53.1-4.53.21. © 2012 by John Wiley & Sons, Inc.
    Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] 12/2012; Chapter 4:Unit4.53.
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    ABSTRACT: The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed "M1.3" as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.
    Nanoscale 11/2012; · 6.23 Impact Factor
  • Angewandte Chemie 08/2012; 124(33).
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    ABSTRACT: Extending both ways: A method for DNA-templated synthesis on solid support is described. Controlled, stepwise chain extension was demonstrated both in the direction favored by nature (3'-extension; see scheme) and in the direction typical for conventional DNA synthesizers (5'-extension).
    Angewandte Chemie International Edition 07/2012; 51(33):8299-303. · 11.34 Impact Factor
  • Heike Vogel, Clemens Richert
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    ABSTRACT: The discovery of small RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi-associated RNAs (piRNAs) has led to new challenges in the selective detection of RNAs. Many noncoding RNAs act as post-translational regulators of gene expression and are involved in the regulation of cell proliferation or apoptosis, but are difficult to amplify, label, and detect. Standard microarray detection procedures involve pre-hybridization labeling or enzymatic 3'-labeling by polymerase-catalyzed extension. Dual labeling would improve the fidelity of detection, but no polymerases for 5'-extension are known. Here we report a novel labeling method for RNAs bearing natural 5'-phosphate groups, such as miRNAs, based on enzyme-free ligation of a biotin- or fluorophore-labeled oligonucleotide to the 5' termini. The method uses in situ activation of the natural 5'-phosphate groups in these RNAs and was optimized to give near-quantitative conversion in solution. With use of biotin- or fluorophore-bearing labeling strands, different miRNA sequences were detected on microarrays with little background fluorescence. In combination with an established method of enzymatic on-chip labeling at the 3' termini, highly selective detection of related miRNAs was achieved by dual recognition at both termini, even in the case of miRNAs differing in only one nucleotide.
    ChemBioChem 06/2012; 13(10):1474-82. · 3.74 Impact Factor
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    ABSTRACT: Branched oligonucleotides with "CG zippers" as DNA arms assemble into materials from micromolar solutions. Their synthesis has been complicated by low yields in solid-phase syntheses. Here we present a solution-phase synthesis based on phosphoramidites of dimers and phenolic cores that produces six-arm or four-arm hybrids in up to 61% yield. On the level of hybrids, only the final product has to be purified by precipitation or chromatography. A total of five different hybrids were prepared via the solution-phase route, including new hybrid (TCG)(4)TTPA with a tetrakis(triazolylphenyl)adamantane core and trimer DNA arms. The new method is more readily scaled up than solid-phase syntheses, uses no more than 4 equiv of phosphoramidite per phenolic alcohol, and provides routine access to novel materials that assemble via predictable base-pairing interactions.
    The Journal of Organic Chemistry 02/2012; 77(6):2703-17. · 4.56 Impact Factor
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    ABSTRACT: A method for the solution-phase synthesis of branched oligonucleotides with tetrahedral or pseudo-octahedral geometry is described that involves the coupling of 3'-H-phosphonates of protected dinucleoside phosphates and organic core molecules. The dimer building blocks are produced by a synthesis that requires no chromatographic purification and that produces the dimer H-phosphonates in up to 44% yield in less than three days of laboratory work. A total of seven different branched hybrids were prepared, including a new hybrid of the sequence (CG)(4)TBA, where TBA stands for tetrakis(p-hydroxybiphenyl)adamantane that assembles into a material from micromolar aqueous solution upon addition of MgCl(2).
    The Journal of Organic Chemistry 02/2012; 77(6):2718-28. · 4.56 Impact Factor
  • J Org Chem. 02/2012;
  • J Org Chem. 02/2012;
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    ABSTRACT: Es geht in beide Richtungen: Ein Festphasen‐Verfahren zur Synthese auf einem DNA‐Templat ermöglicht die schrittweise Kettenverlängerung sowohl in die von der Natur bevorzugte Richtung (3′‐Extension; siehe Schema) als auch in die für gängige DNA‐Synthesizer typische Richtung (5′‐Extension).
    Angewandte Chemie 01/2012; 124(33).

Publication Stats

652 Citations
604.60 Total Impact Points

Institutions

  • 2009–2014
    • Universität Stuttgart
      • Institute of Organic Chemistry
      Stuttgart, Baden-Württemberg, Germany
  • 2003–2010
    • Karlsruhe Institute of Technology
      • Institute of Organic Chemistry
      Karlsruhe, Baden-Wuerttemberg, Germany
  • 2001–2010
    • Universität Konstanz
      • Department of Chemistry
      Constance, Baden-Württemberg, Germany
  • 1997–2010
    • Tufts University
      • Department of Chemistry
      Georgia, United States
  • 2005
    • Max Planck Institute for Biophysical Chemistry
      Göttingen, Lower Saxony, Germany
  • 1993–1997
    • Ludwig-Maximilian-University of Munich
      München, Bavaria, Germany
  • 1996
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland
  • 1994
    • University of Cologne
      • Institute of Organic Chemistry
      Köln, North Rhine-Westphalia, Germany