M Sprinzl

University of Bayreuth , Bayreuth, Bavaria, Germany

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Publications (235)1004.8 Total impact

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    ABSTRACT: Existing technologies for analysis of microbiological contaminants in food or clinical samples are often expensive and require laboratory settings and trained personnel. Here we present a lateral flow assay employing gold nanoparticle-oligodeoxynucleotide conjugates and four-component sandwich hybridisation for direct detection of specific sequences in bacterial 16S ribosomal RNA. Combined with rapid "one step" lysis the developed procedure allows detection of 5 × 10(4) colony forming units per mL Escherichia coli within less than 25 minutes. Several Escherichia coli strains were detected successfully, whereas non-related as well as closely related bacterial species produced no signal. The developed nucleic acid lateral flow assay is inexpensive, rapid to perform and requires no nucleic acid amplification step.
    The Analyst 01/2014; · 4.23 Impact Factor
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    ABSTRACT: Naturally occurring nucleotide modifications within RNA have been proposed to be structural determinants for innate immune recognition. We tested this hypothesis in the context of native nonself-RNAs. Isolated, fully modified native bacterial transfer RNAs (tRNAs) induced significant secretion of IFN-α from human peripheral blood mononuclear cells in a manner dependent on TLR7 and plasmacytoid dendritic cells. As a notable exception, tRNA(Tyr) from Escherichia coli was not immunostimulatory, as were all tested eukaryotic tRNAs. However, the unmodified, 5'-unphosphorylated in vitro transcript of tRNA(Tyr) induced IFN-α, thus revealing posttranscriptional modifications as a factor suppressing immunostimulation. Using a molecular surgery approach based on catalytic DNA, a panel of tRNA(Tyr) variants featuring differential modification patterns was examined. Out of seven modifications present in this tRNA, 2'-O-methylated G(m)18 was identified as necessary and sufficient to suppress immunostimulation. Transplantation of this modification into the scaffold of yeast tRNA(Phe) also resulted in blocked immunostimulation. Moreover, an RNA preparation of an E. coli trmH mutant that lacks G(m)18 2'-O-methyltransferase activity was significantly more stimulatory than the wild-type sample. The experiments identify the single methyl group on the 2'-oxygen of G(m)18 as a natural modification in native tRNA that, beyond its primary structural role, has acquired a secondary function as an antagonist of TLR7.
    Journal of Experimental Medicine 02/2012; 209(2):225-33. · 13.21 Impact Factor
  • Yiwei Huang, Mathias Sprinzl
    Angewandte Chemie 08/2011; 123(32).
  • Yiwei Huang, Mathias Sprinzl
    Angewandte Chemie International Edition 03/2011; 50(32):7287-9. · 11.34 Impact Factor
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    ABSTRACT: Detection of foodborne pathogenic and spoilage bacteria by RNA-DNA hybridization is an alternative to traditional microbiological procedures. To achieve high sensitivity with RNA-DNA-based methods, efficient bacterial lysis and release of nucleic acids from bacteria are needed. Here we report the specific detection of the hygiene indicator microorganism Escherichia coli in meat by use of electrochemical biochips. We improved RNA isolation from bacteria in meat juice from pork and beef. Samples, either naturally or artificially contaminated by E. coli, were enriched by incubation in full or minimal medium. A combined treatment of the samples with lysozyme, proteinase K, and sonication resulted in efficient cell disruption and high total RNA yields. Together with optimization of enrichment time, this ensures high sensitivity of electrochemical measurements on biochips. A short enrichment period and the triple-lysis regimen in combination with electrochemical biochip measurement were tested with 25 meat samples. The lower limit of detection of the biochip was approximately 2,000 CFU of E. coli per ml. The entire analysis procedure (5 h of enrichment, triple lysis, and biochip detection) has a lower limit of detection of 1 CFU of E. coli per ml within a total time needed for analysis of 7 h.
    Journal of food protection 11/2010; 73(11):2025-33. · 1.83 Impact Factor
  • Christopher Pöhlmann, Mathias Sprinzl
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    ABSTRACT: MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.
    Analytical Chemistry 06/2010; 82(11):4434-40. · 5.82 Impact Factor
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2010; 26(2).
  • Fleischwirtschaft -Frankfurt- 01/2010; 90(7):96-99. · 0.11 Impact Factor
  • Mathias Sprinzl, Volker A Erdmann
    ChemBioChem 11/2009; 10(18):2851-3. · 3.74 Impact Factor
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    ABSTRACT: A method is presented by which an azide-containing side chain can be introduced into any internal position of a polypeptide chain by in vitro translation. For this, 2'-deoxy-cytidylyl-(3'-->5')-adenosine was acylated on the 3'(2')-hydroxyl group of adenosine with 6-azido-2(S)-hydroxyhexanoic acid (AHHA), an alpha-hydroxy- and epsilon-azide derivative of L-lysine. The acylated dinucleotide was enzymatically ligated with a tRNA transcript to provide chemically stable E. coli suppressor AHHA-tRNA(Cys(CUA)). The esterase 2 gene from Alicyclobacillus acidocaldarius was modified by the amber stop codon (UAG) on position 118. Using AHHA-tRNA(Cys(CUA)) in an E. coli in vitro translation/transcription system, the site-directed introduction of an azide group linked to a backbone ester into the esterase polypeptide was achieved. The yield of the synthesized modified protein reached 80% compared to translation of the native esterase. Subsequently, azide coupling with an alkyne-modified oligodeoxynucleotide demonstrated the feasibility of this approach for conjugation of polypeptides.
    Organic & Biomolecular Chemistry 10/2009; 7(20):4218-24. · 3.57 Impact Factor
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    ABSTRACT: A method is presented by which an azide-containing side chain can be introduced into any internal position of a polypeptide chain by in vitro translation. For this, 2′-deoxy-cytidylyl-(3′→5′)-adenosine was acylated on the 3′(2′)-hydroxyl group of adenosine with 6-azido-2(S)-hydroxyhexanoic acid (AHHA), an α-hydroxy- and ε-azide derivative of L-lysine. The acylated dinucleotide was enzymatically ligated with a tRNA transcript to provide chemically stable E. coli suppressor AHHA-tRNACys(CUA). The esterase 2 gene from Alicyclobacillus acidocaldarius was modified by the amber stop codon (UAG) on position 118. Using AHHA-tRNACys(CUA) in an E. coli in vitro translation/transcription system, the site-directed introduction of an azide group linked to a backbone ester into the esterase polypeptide was achieved. The yield of the synthesized modified protein reached 80% compared to translation of the native esterase. Subsequently, azide coupling with an alkyne-modified oligodeoxynucleotide demonstrated the feasibility of this approach for conjugation of polypeptides.
    Organic & Biomolecular Chemistry 09/2009; 7(20). · 3.57 Impact Factor
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    ABSTRACT: The lOSa RNA gene of Thermus thermophilus was isolated and sequenced. The tRNA-like structure at the 5′ and 3′ ends and other secondary structure features of the T. thermophilus l0Sa RNA are similar to E. coli l0Sa RNA. A variant of the sequence motif coding for the tag peptide is located in the centre of T. thermophilus l0Sa RNA.
    Mitochondrial DNA 07/2009; 9(1):31-35. · 1.71 Impact Factor
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    ABSTRACT: Novel enzyme-oligodeoxynucleotide conjugate was synthesized to improve sensitivity of Escherichia coli 16S rRNA detection on gold electrodes. Thermostable esterase 2 from Alicyclobacillus acidocaldarius was multiply conjugated to a polyamidoamine dendrimer functionalized by one universal detector oligodeoxynucleotide. Three components rRNA/DNA hybridization between capture oligodeoxynucleotide covalently immobilized on a gold electrode, 16S rRNA and the multivalent esterase-dendrimer cluster was used for detection of E. coli. The linear dependence of the electrochemical signals to analyte concentration revealed a detection limit of 50 colony forming units E. coli, which represents a tenfold signal enhancement if compared to the detection limit achieved with monovalent esterase-oligodeoxynucleotide conjugate.
    Biosensors & Bioelectronics 05/2009; 24(11):3383-6. · 6.45 Impact Factor
  • Mathias Sprinzl
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    ABSTRACT: Quality control: The incorporation of a wrong amino acid into a growing polypeptide chain induces a correction step in which the release factor (RF1) hydrolyzes the peptide from the incorrectly matched peptidyl-tRNA (see picture). The nascent erroneous polypeptide is released from the ribosome and degraded.
    Angewandte Chemie International Edition 04/2009; 48(21):3738-9. · 11.34 Impact Factor
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    ABSTRACT: Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2-ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two gram-negative and two gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination.
    Biosensors & Bioelectronics 03/2009; 24(9):2766-71. · 6.45 Impact Factor
  • Mathias Sprinzl
    Angewandte Chemie 01/2009; 121(21):3792-3793.
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    ABSTRACT: 5'-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterase-catalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3-and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.
    Bioconjugate Chemistry 12/2008; 19(12):2456-61. · 4.58 Impact Factor
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    ABSTRACT: One of the first specialized collections of nucleic acid sequences in life sciences was the 'compilation of tRNA sequences and sequences of tRNA genes' (http://www.trna.uni-bayreuth.de). Here, an updated and completely restructured version of this compilation is presented (http://trnadb.bioinf.uni-leipzig.de). The new database, tRNAdb, is hosted and maintained in cooperation between the universities of Leipzig, Marburg, and Strasbourg. Reimplemented as a relational database, tRNAdb will be updated periodically and is searchable in a highly flexible and user-friendly way. Currently, it contains more than 12 000 tRNA genes, classified into families according to amino acid specificity. Furthermore, the implementation of the NCBI taxonomy tree facilitates phylogeny-related queries. The database provides various services including graphical representations of tRNA secondary structures, a customizable output of aligned or un-aligned sequences with a variety of individual and combinable search criteria, as well as the construction of consensus sequences for any selected set of tRNAs.
    Nucleic Acids Research 11/2008; 37(Database issue):D159-62. · 8.81 Impact Factor
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    ABSTRACT: His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF-Tu, [H85L] EF-Tu and [H85G]EF-Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF-Tu, and replacement of Arg300 provided [R300I]EF-Tu. Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF-Tu · GTP, inducing a protease-sensitive cleavage site in domain II. [H85Q]EF-TU, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes. The slow GTPase of [H85Q]EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation. Replacement of His85 in T. thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding. [H85L]EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis. The rate of nucleotide dissociation is highest for [H85L]EF-Tu, followed by [H85Q]EF-Tu and native T. thermophilus EF-Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.
    European Journal of Biochemistry. 06/2008; 229(3):596 - 604.
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    ABSTRACT: Recently, we have shown that anions of Hofmeister series affect the enzyme activity through modulation of flexibility of its active site. The enzyme activity vs. anion position in Hofmeister series showed an unusual bell-shaped dependence. In the present work, six monovalent cations (Na(+), Gdm(+), NH(4)(+), Li(+), K(+) and Cs(+)) of Hofmeister series with chloride as a counterion have been studied in relation to activity and stability of flavoprotein NADH oxidase from Thermus thermophilus (NOX). With the exception of strongly chaotropic guanidinium cation, cations are significantly less effective in promoting the Hofmeister effect than anions mainly due to repulsive interactions of positive charges around the active site. Thermal denaturations of NOX reveal unfavorable electrostatic interaction at the protein surface that may be shielded to different extent by salts. Michaelis-Menten constants for NADH, accessibility of the active site as reflected by Stern-Volmer constants and activity of NOX at high cation concentrations (1-2 M) show bell-shaped dependences on cation position in Hofmeister series. Our analysis indicates that in the presence of kosmotropic cations the enzyme is more stable and possibly more rigid than in the presence of chaotropic cations. Molecular dynamic (MD) simulations of NOX showed that active site switches between open and closed conformations [J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465-476]. Enzyme activity, as well as substrate binding, can be regulated by the salt mediated perturbation of the balance between open and closed forms. We propose that compensating effect of accessibility and flexibility of the enzyme active site leads to bell-shaped dependence of the investigated parameters.
    Biochimica et Biophysica Acta 06/2008; 1784(5):789-95. · 4.66 Impact Factor

Publication Stats

6k Citations
1,004.80 Total Impact Points

Institutions

  • 1980–2011
    • University of Bayreuth
      • Chair of Biochemistry
      Bayreuth, Bavaria, Germany
  • 2008
    • Tomas Bata University in Zlín
      • Department of Physics and Materials Engineering
      Zlín, Zlinsky kraj, Czech Republic
  • 2001–2008
    • Pavol Jozef Šafárik University in Košice
      • Department of Biochemistry
      Košice, Kosicky Kraj, Slovakia
  • 1972–2005
    • Max Planck Institute for Experimental Medicine
      Göttingen, Lower Saxony, Germany
  • 2004
    • Philipps-Universität Marburg
      • Institut für Pharmazeutische Chemie
      Marburg an der Lahn, Hesse, Germany
  • 2002
    • Parazitologický ústav SAV
      Presburg, Bratislavský, Slovakia
  • 1999–2000
    • Max-Delbrück-Centrum für Molekulare Medizin
      Berlín, Berlin, Germany
  • 1996
    • Universität Konstanz
      Constance, Baden-Württemberg, Germany
  • 1992
    • Brandeis University
      Waltham, Massachusetts, United States
  • 1991
    • Academy of Sciences of the Czech Republic
      Praha, Praha, Czech Republic
  • 1977
    • Hebrew University of Jerusalem
      • Department of Biological Chemistry
      Yerushalayim, Jerusalem District, Israel
  • 1974
    • Max Planck Institute for Chemistry
      Mayence, Rheinland-Pfalz, Germany