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M Famulok

Publications and source records attributed to M Famulok.

At least 37 records · Page 2Linked to original sources
PubMed1998

Design and synthesis of a transition state analogue for the Diels-Alder reaction.

This paper describes the design and synthesis of a tricationic transition state analogue (TSA 1) for the Diels-Alder reaction. TSA 1 contains a bicyclo[2.2.1]heptene ring system that mimics the boat conformation of the Diels-Alder transition state and is designed to bind tightly to antibodies, nucleic acids, and imprinted polymers by means of hydrogen bonds and salt-bridges. This paper also describes the syntheses of the Diels-Alder reaction substrates (diene 2 and dienophile 3) and a sensitive HPLC assay to monitor the formation of Diels-Alder product 4. In contrast to previously reported TSAs and dienophiles for the Diels-Alder reaction that are based upon maleimides, TSA 1 and dienophile 3 are based upon fumaramide. The fumaramide system should destabilize the initially formed boat conformer of Diels-Alder product 4 and stabilize a half-chair conformer. The conversion of the initially formed boat conformer to the half-chair conformer is designed to help prevent Diels-Alder product 4 from binding strongly to catalysts selected to strongly bind TSA 1. This feature should minimize product inhibition, which can be a problem in the catalysis of the Diels-Alder reaction.

Bridged Bicyclo Compounds↗
PubMed1998

A novel ribozyme with ester transferase activity.

BACKGROUND: The 'RNA world' hypothesis proposes that the early history of life on earth consisted of a period in which chemical transformations were catalyzed exclusively by ribozymes. Ribozymes that act as acyl transferases, or catalyze the formation of amide or peptide bonds seem particularly attractive candidates to link the RNA world to the modern protein-nucleic acid world. The central role played by aminoacylated RNAs in today's processes of translating RNA into protein suggests that aminoacyl transfer reactions catalyzed by RNA might have facilitated the development and optimization of the translation apparatus during early evolution. RESULTS: We describe the isolation and characterization of a novel ribozyme that catalyzes the transfer of an amino-acid ester from an aminoacyl donor substrate onto the ribozyme itself. The site of aminoacylation was determined to be at an internal 2'-OH group of a cytosine residue. The aminoacylation depends on the presence of Mg2+ and can be competitively inhibited by the AMP moiety of the aminoacyl donor substrate, suggesting that there is a specific binding pocket for this substrate. The originally selected ribozyme was engineered to act as intermolecular catalyst that transfers the amino acid onto an external 28-residue oligonucleotide. The aminoacylated oligonucleotide was further used to quantify the reverse reaction catalyzed by the ribozyme. CONCLUSIONS: The ribozyme we have isolated is an example of a catalytic RNA with ester transferase activity which uses a substrate that is not templated by Watson-Crick-pairing hydrogen bonds. The reaction catalyzed by the ribozyme expands the scope of RNA catalysis to include acyl transferase activity from an RNA 3' end to an internal 2' position and the reverse. Ribozymes with such activity have been postulated to be evolutionary precursors of ribosomal RNA.

Acyltransferases↗
PubMed1998

Oligonucleotide libraries--variatio delectat.

In vitro selection of combinatorial nucleic acid libraries leads to specific target-binding molecules--RNA, single stranded DNA, modified RNA or modified DNA, commonly designated as aptamers--and to novel catalytic nucleic acids. The current state of aptamer and ribozyme technology is such that it establishes itself as a means of obtaining useful tools for molecular biology, diagnostics, molecular medicine and bio-organic chemistry.

Base Sequence↗
PubMed1997

Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers.

To probe the complex nucleic acid binding domains of yeast RNA polymerase II (Pol II), we have isolated in the presence of heparin RNA molecules that selectively bind to yeast Pol II. A class of RNA molecules was found to bind and strongly interfere with enzyme-DNA interaction but not with RNA chain elongation. Remarkably, one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II. S. cerevisiae Pol I and Pol III and Pol II from Schizosaccharomyces pombe or wheat germ cells were not affected. Photocross-linking experiments showed that the RNA ligand preferentially interacted with B220, the largest subunit of Pol II and, to a lesser extent, with B150, the second largest subunit. The selected RNA was expressed in yeast cells under the control of a Pol III promoter. Yeast cells that expressed the anti-Pol II aptamer grew normally. However, a cell growth defect was observed when expressing the RNA aptamer in cells having an artificially reduced level of Pol II.

Base Sequence↗
PubMed1997

Isoalloxazine derivatives promote photocleavage of natural RNAs at G.U base pairs embedded within helices.

We have recently shown that isoalloxazine derivatives are able to photocleave RNA specifically at G.U base pairs embedded within a helical stack. The reaction involves the selective molecular recognition of G.U base pairs by the isoalloxazine ring and the removal of one nucleoside downstream of the uracil residue. Divalent metal ions are absolutely required for cleavage. Here we extend our studies to complex natural RNA molecules with known secondary and tertiary structures, such as tRNAs and a group I intron (td). G.U pairs were cleaved in accordance with the phylogenetically and experimentally derived secondary and tertiary structures. Tandem G.U pairs or certain G.U pairs located at a helix extremity were not affected. These new cleavage data, together with the RNA crystal structure, allowed us to perform molecular dynamics simulations to provide a structural basis for the observed specificity. We present a stable structural model for the ternary complex of the G. U-containing helical stack, the isoalloxazine molecule and a metal ion. This model provides significant new insight into several aspects of the cleavage phenomenon, mechanism and specificity for G. U pairs. Our study shows that in large natural RNAs a secondary structure motif made of an unusual base pair can be recognized and cleaved with high specificity by a low molecular weight molecule. This photocleavage reaction thus opens up the possibility of probing the accessibility of G.U base pairs, which are endowed with specific structural and functional roles in numerous structured and catalytic RNAs and interactions of RNA with proteins, in folded RNAs.

Base Composition↗
PubMed1997

In vitro and in vivo characterization of novel mRNA motifs that bind special elongation factor SelB.

The special elongation factor SelB of Escherichia coli promotes selenocysteine incorporation into formate dehydrogenases. This is thought to be achieved through simultaneous binding to selenocysteyl-tRNASec and, in the case of formate dehydrogenase H, to an fdhF mRNA hairpin structure 3' adjacent to the UGA selenocysteine codon. By in vitro selection, novel RNA sequences ("aptamers"), which can interact tightly and specifically with SelB, were isolated from an RNA library. The library was comprised of mutagenized variants of the wild-type fdhF mRNA hairpin. One-half of the selected sequences contained the apical stem-loop of the fdhF mRNA hairpin highly conserved. Some of the aptamers showed deviations in the primary sequence within this region of the wild-type fdhF hairpin motif while still binding with high affinity to SelB. Binding studies performed with truncated versions of SelB revealed that aptamers binding to different sites on the protein have been selected. To dissect SelB binding to the fdhF hairpin from the overall biological function of this complex, four selected aptamers were analyzed in vivo for UGA readthrough in a lacZ fusion construct. Among these, one promoted UGA readthrough in vivo. Three of the aptamers, however, were drastically reduced or unable to replace the fdhF mRNA hairpin in vivo, despite the similar secondary structure and binding affinities of these RNAs compared with the wild-type motif. This finding implies functions of the fdhF hairpin that go beyond the mere tethering of selenocysteyl-tRNASec to the UGA codon.

Bacterial Proteins↗
PubMed1997

Characterization and divalent metal-ion dependence of in vitro selected deoxyribozymes which cleave DNA/RNA chimeric oligonucleotides.

By in vitro selection, a variety of catalytic DNA oligonucleotides were obtained which cleave chimeric oligonucleotides at a single ribonucleotide position embedded within a deoxyribonucleotide context in the presence or absence of divalent metal ions. After several cycles of selection/amplification in the absence and in the presence of low amounts of Mg2+ two different types of catalysts emerged: one type depended strongly on Mg2+ or other divalent metal ions, the other type performed cleavage reactions independently of Mg2+ in the presence of spermine. Experimental analysis of the secondary structure of some of the selected deoxyribozymes was carried out by chemical probing. The ribonucleotide in the selected catalysts is unpaired and presents the cleavage site to the attacking nucleophile. Our results suggest that the main selection criterion under metal-free conditions was a favourable arrangement of the attacking nucleophile and the phosphate leaving group. The cleavage rates of the selected divalent metal independent catalysts are within the same order of magnitude as the rate of metal independent substrate hydrolysis in the hammerhead ribozyme. One of the metal dependent catalysts showed an unexpected preference for Ca2+ instead of Mg2+. In this deoxyribozyme binding of Ca2+ occurred co-operatively whereas binding of Mg2+ did not. Comparison of the secondary structure and reactivity of this catalyst with Mg2+ and Ca2+ suggests that here a special binding pocket for Ca2+ was selected. This deoxyribozyme achieved a rate acceleration of substrate cleavage in the order of at least 10(4) compared to the uncatalysed reaction performing a cleavage mechanism similar to that of the hammerhead or hairpin ribozyme.

Allosteric Regulation↗
PubMed1997

In vitro selection of a viomycin-binding RNA pseudoknot.

BACKGROUND: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection. RESULTS: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot. CONCLUSIONS: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.

Anti-Bacterial Agents↗
PubMed1997

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.

Animals↗
PubMed1996

Structural basis of ligand discrimination by two related RNA aptamers resolved by NMR spectroscopy.

In a previous study, an RNA aptamer for the specific recognition of arginine was evolved from a parent sequence that bound citrulline specifically. The two RNAs differ at only 3 positions out of 44. The solution structures of the two aptamers complexed to their cognate amino acids have now been determined by two-dimensional nuclear magnetic resonance spectroscopy. Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA but that fold into a compact structure upon ligand binding. Those nucleotides common to both RNAs include a conserved cluster of purine residues, three of which form an uneven plane containing a G:G pair, and two other residues nearly perpendicular to that surface. Two of the three variant nucleotides are stacked on the cluster of purines and form a triple contact to the amino acid side chain, whereas the edge of the third variant nucleotide is capping the binding pocket.

Arginine↗
PubMed1996

In vitro selection analysis of neomycin binding RNAs with a mutagenized pool of variants of the 16S rRNA decoding region.

An in vitro selection for neomycin B binding was carried out with an RNA pool containing a 47-nucleotide domain of the decoding region of 16S ribosomal RNA, mutated at 30% per base position. The degenerate region was comprised of an oligonucleotide analogue ("motif A") of the decoding region in 30S subunits which has previously been shown to interact with the aminoglycoside antibiotic neomycin B and tRNA ligands. After five cycles of selection/amplification, RNA sequences were isolated which specifically bound to neomycin B. Cloning and sequencing showed that none of the isolated clones shared primary sequence or secondary structure homology with the decoding region of 16S RNA. Instead, a new set of sequences was isolated which could be folded into a defined hairpin structure designated as motif B. We investigated the affinity of motif A, motif B, the unselected pool RNA, and the corresponding unmutagenized "parent" RNA to neomycin B at different Mg2+ concentrations. Under buffer conditions of low ionic strength all RNAs tested bound nonspecifically to neomycin B. However, motif B bound to neomycin B at Mg2+ concentrations at which binding of the other RNAs tested was significantly lower or not detectable. This is consistent with motif B exhibiting a higher affinity for neomycin B than motif A under these conditions. Motif B has previously been isolated from an in vitro selection to identify RNA sequences with affinity to neomycin B using a completely randomized RNA pool which shared no relationship to motif A. Our results indicate that motif B might represent a highly optimized RNA sequence for neomycin B binding; conversely, the A-site motif in 16S rRNA (motif A) might not be an optimal target for neomycin B recognition.

Anti-Bacterial Agents↗
PubMed1996

RNA aptamers that bind L-arginine with sub-micromolar dissociation constants and high enantioselectivity.

A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 0 muM were used to select for tight binding arginine specific RNA aptamers. A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders. The selection scheme included a negative selection with the non-cognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine. After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid. When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column. These RNA aptamers were cloned and sequenced. Equilibrium dialysis performed with the most abundant clone among the selected sequence revealed Kd values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date. Arginine recognition by this RNA is highly enantioselectice: L- arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif. Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine. The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments.

Animals↗
PubMed1996

Prion protein PrPc interacts with molecular chaperones of the Hsp60 family.

Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans. The prion particle consists mainly, if not entirely, of PrPSc, a posttranslationally modified isoform of the cellular host-encoded prion protein (PrPc). It has been suggested that additional cellular factors might be involved in the physiological function of PrPc and in the propagation of PrPSc. Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrPc. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.

Animals↗
PubMed1995

Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding.

In a recent study, an RNA aptamer for the specific recognition of the amino acid L-arginine was evolved from an in vitro selected L-citrulline binding parent sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1698-1706]. We have now carried out a structural analysis of these aptamers by using chemical modification experiments. Footprinting experiments and a damage selection approach were performed to identify those positions protected from modification in the presence of the amino acids and modifications that interfere with the binding of the ligand. It is shown that of the two bulged regions present in both aptamers one can be modified without loss of binding activity whereas in the other bulge nearly every position is shown to be involved in the recognition of the ligands. This might be indicative for non-canonical base pairing to occur within the non-Watson-Crick paired regions which might be stabilized by the complexed amino acid. Binding to the cognate amino acid significantly enhances the conformational stability of the RNA. We also tested the sensitivity of both aptamers towards lead (II) ion induced cleavage and identified a hypersensitive cleavage site within the invariant bulged region. Lead cleavage is inhibited by the complexed amino acid, indicating a conformational change of the aptamer upon ligand binding. NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indicate the formation of a 1:1 complex of RNA:amino acid.

Arginine↗
PubMed1995

A novel RNA motif for neomycin recognition.

BACKGROUND: Antibiotics can interfere with RNA activity. Translation of RNA by the prokaryotic ribosome, self-splicing of group I introns, HIV replication and hammerhead ribozyme cleavage are inhibited by the aminoglycoside neomycin B. To explore the molecular basis by which small molecules such as antibiotics inhibit RNA function, we undertook an in vitro selection to obtain a variety of RNA molecules with the capacity to recognize neomycin. RESULTS: The majority of the RNA molecules selected to specifically bind neomycin share a region of nucleotide sequence homology. From chemical probing and covariations among different clones we show that in all sequences this region folds into a hairpin structure, which from footprinting and partial alkaline hydrolysis experiments is shown to be the neomycin-binding site. Neomycin is recognized with high affinity (Kd approximately equal to 100 nM) and high specificity (> 100-fold higher affinity for neomycin than for paromomycin). CONCLUSIONS: The fact that RNAs containing the consensus sequence, as well as sequences that display variations within this region, specifically recognize neomycin suggests that a structural motif rather than a particular nucleotide sequence is required for neomycin recognition. We propose that a hairpin stem-loop structural motif, which might feature a widened major groove, may be a prerequisite for neomycin recognition. This structural pattern can be extrapolated to other natural neomycin-responsive RNAs.

Anti-Bacterial Agents↗
PubMed1995

Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems.

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.

Animals↗
PubMed1994

All you wanted to know about SELEX.

In vitro selection, or SELEX, is a technique that allows the simultaneous screening of highly diverse pools of different RNA or DNA (dsDNA or ssDNA) molecules for a particular feature. Different examples from a great variety of applications of in vitro selection experiments are described and a detailed overview of the method and its variations will be given. Some especially conclusive in vitro selection experiments are discussed in detail to illustrate the potential power and diversity of this method. Potential restrictions of the methods and possible ways to overcome them are pointed out.

Base Sequence↗