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PubMed2001

Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins.

RNA affinity tags would be very useful for the study of RNAs and ribonucleoproteins (RNPs) as a means for rapid detection, immobilization, and purification. To develop a new affinity tag, streptavidin-binding RNA ligands, termed "aptamers," were identified from a random RNA library using in vitro selection. Individual aptamers were classified into two groups based on common sequences, and representative members of the groups had sufficiently low dissociation constants to suggest they would be useful affinity tools. Binding of the aptamers to streptavidin was blocked by presaturation of the streptavidin with biotin, and biotin could be used to dissociate RNA/streptavidin complexes. To investigate the practicality of using the aptamer as an affinity tag, one of the higher affinity aptamers was inserted into RPR1 RNA, the large RNA subunit of RNase P. The aptamer-tagged RNase P could be specifically isolated using commercially available streptavidin-agarose and recovered in a catalytically active form when biotin was used as an eluting agent under mild conditions. The aptamer tag was also used to demonstrate that RNase P exists in a monomeric form, and is not tightly associated with RNase MRP, a closely related ribonucleoprotein enzyme. These results show that the streptavidin aptamers are potentially powerful tools for the study of RNAs or RNPs.

Affinity Labels↗
PubMed2001

Electronically transduced molecular mechanical and information functions on surfaces.

Supramolecular chemistry and nanotechnology, along with their use in the construction of functional assemblies and devices, have merged into a challenging field of study. The development of methodologies for the integration and interfacing of molecular building blocks with solid supports and electronic transducers is essential for this research. We address recent applications of molecular, macromolecular, and biomolecular substances in the organization of signal-activated, electronically transduced molecular architectures on electrode surfaces. Photonic, electronic, magnetic, and chemical stimuli are used to trigger the switchable functions of these systems, which demonstrate either mechanical (e.g., translocation) or computational (e.g., memory) functions and provide enlightening insight and directions for the future evolution of the field.

Electrodes↗
PubMed2005

Relationship between folding and function in a sequence-specific miniature DNA-binding protein.

Previously, we have described a miniature protein-based approach to the design of molecules that bind DNA or protein surfaces with high affinity and specificity. In this approach, the small, well-folded protein avian pancreatic polypeptide acts as a scaffold to present and stabilize an alpha-helical or PPII-helical recognition epitope. The first miniature protein designed in this way, a molecule called p007, presents the alpha-helical recognition epitope found on the bZIP protein GCN4 and binds DNA with nanomolar affinity and exceptional specificity. In this work we use alanine-scanning mutagenesis to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. Virtually every residue within the p007 alpha-helix, and most residues within the p007 PPII helix, contribute to both DNA affinity and specificity. These residues include those introduced to make specific and nonspecific DNA contacts, as well as those that complete the miniature protein core. Moreover, there exists a direct correlation between the affinity of a p007 variant for specific DNA and the ability of that variant to select for specific DNA over nonspecific DNA. Although we observe no correlation between alpha-helicity and affinity, we observe a limited correlation between alpha-helicity and sequence specificity that emphasizes the role of coupled binding/folding in the function of p007. Our results imply that formation of a highly evolved set of protein.DNA contacts in the context of a well-packed hydrophobic core, and not the extent of intrinsic alpha-helical structure, is the primary determinant of p007 function.

Amino Acid Sequence↗
PubMed2005

Discovery of acetylcholinesterase peripheral anionic site ligands through computational refinement of a directed library.

The formation of beta-amyloid plaques in the brain is a key neurodegenerative event in Alzheimer's disease. Small molecules capable of binding to the peripheral anionic site of acetylcholinesterase (AChE) have been shown to inhibit the AChE-induced aggregation of the beta-amyloid peptide. Using the combination of a computational docking model and experimental screening, five compounds that completely blocked the amyloidogenic effect of AChE were rapidly identified from an approximately 200-member library of compounds designed to disrupt protein-protein interactions. Critical to this docking model was the inclusion of two explicit water molecules that are tightly bound to the enzyme. Interestingly, none of the tested compounds inhibited the related enzyme butyrylcholinesterase (BuChE) up to their aqueous solubility limits. These compounds are among the most potent inhibitors of amyloid beta-peptide aggregation and are equivalent only to propidium, a well-characterized AChE peripheral anionic site binder and aggregation inhibitor.

Acetylcholinesterase↗
PubMed1996

Targeting sites within HIV-1 cDNA with a DNA-cleaving ribozyme.

A variant of the Tetrahymena ribozyme that efficiently cleaves single-stranded DNA under simulated physiological conditions [Tsang, J., & Joyce, G. F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potential therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites within HIV-I cDNA. In order to increase the sequence selectivity of the ribozyme, its substrate recognition domain was extended from 6 to 12 nucleotides, allowing base pairing with substrate nucleotides that lie both upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a variety of different DNA targets. The ribozyme exhibited a high degree of sequence specificity, discriminating by a factor of 10(2) to more than 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the cleavage site led to a greater decrease in activity compared to those that occurred farther away.

Animals↗
PubMedDate not supplied

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification.

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.

Bacterial Proteins↗
PubMed2001

Enzyme redesign.

Explore the source record for details and available documents.

Adenosine Triphosphate↗
PubMed1992

Nonenzymatic template-directed synthesis on hairpin oligonucleotides. 2. Templates containing cytidine and guanosine residues.

We have prepared hairpin oligonucleotides in which a 5'-terminal single-stranded segment contains cytidylate (C) and guanylate (G) residues. When these hairpin substrates are incubated with a mixture of cytidine 5'-phosphoro(2-methly)imidazolide (2-MeImpC) and guanosine 5'-phosphoro(2-methyl)imidazolide (2-MeImpG), the 5'-terminal segment acts as a template to facilitate sequence-specific addition of G and C residues to the 3'-terminus of the hairpin. If an isolated G residue is present at the 3'-end of the template strand, it is copied regiospecifically in the presence of 2-MeImpC and 2-MeImpG to give a product containing an isolated C residue linked to its G neighbors by 3'-5'-internucleotide bonds. However, if only 2-MeImpC is present in the reaction mixture, very little reaction occurs. Thus, the presence of 2-MeImpG catalyzes the incorporation of C. If the template strand contains a short sequence of G residues, it is copied in the presence of a mixture of 2-MeImpC and 2-MeImpG. If only 2-MeImpC is present in the reaction mixture, efficient synthesis occurs to give a final product containing one fewer C residue than the number of G residues in the template.

Base Sequence↗
PubMed1993

Kinetic dissection of individual steps in the poly(C)-directed oligoguanylate synthesis from guanosine 5'-monophosphate 2-methylimidazolide.

A kinetic study of oligoguanylate synthesis on a polycytidylate template, poly(C), as a function of the concentration of the activated monomer, guanosine 5'-monophosphate 2-methylimidazolide, 2-MeImpG, is reported. Reactions were run with 0.005-0.045 M 2-MeImpG in the presence of 0.05 M poly(C) at 23 degrees C. The kinetic results are consistent with a reaction scheme (eq 1) that consists of a series of consecutive steps, each step representing the addition of one molecule of 2-MeImpG to the growing oligomer. This scheme allows the calculation of second-order rate constants for every step by analyzing the time-dependent growth of each oligomer. Computer simulations of the course of reaction based on the determined rate constants and eq 1 are in excellent agreement with the product distributions seen in the HPLC profiles. In accord with an earlier study (Fakhrai, H.; Inoue, T.; Orgel, L. E. Tetrahedron 1984, 40, 39), rate constants, ki, for the formation of the tetramer and longer oligomers up to the 16-mer were found to be independent of length and somewhat higher than k3 (formation of trimer), which in turn is much higher than k2 (formation of dimer). The ki (i > or = 4), k3, and k2 values are not true second-order rate constants but vary with monomer concentration. Mechanistic models for the dimerization (Scheme I) and elongation reactions (Scheme II) are proposed that are consistent with our results. These models take into account that the monomer associates with the template in a cooperative manner. Our kinetic analysis allowed the determination of rate constants for the elementary processes of covalent bond formation between two monomers (dimerization) and between an oligomer and a monomer (elongation) on the template. A major conclusion from our study is that bond formation between two monomer units or between a primer and a monomer is assisted by the presence of additional next-neighbor monomer units. This is consistent with recent findings with hairpin oligonucleotides (Wu, T.; Orgel, L. E. J. Am. Chem. Soc. 1992, 114, 317). Our study is the first of its kind that shows the feasibility of a thorough kinetic analysis of a template-directed oligomerization and provides a detailed mechanistic model of these reactions.

Chromatography, High Pressure Liquid↗
PubMed2006

A genetically encoded fluorescent amino acid.

The fluorescent amino acid l-(7-hydroxycoumarin-4-yl) ethylglycine 1 has been genetically encoded in E. coli in response to the amber TAG codon. Because of its high fluorescence quantum yield, relatively large Stoke's shift, and sensitivity to both pH and polarity, this amino acid should provide a useful probe of protein localization and trafficking, protein conformation changes, and protein-protein interactions.

Amino Acids↗
PubMed1997

Template-directed synthesis using the heterogeneous templates produced by montmorillonite catalysis. A possible bridge between the prebiotic and RNA worlds.

The synthesis of oligoguanylates [oligo(G)s] is catalyzed by a template of oligocytidylates [oligo(C)s] containing 2',5'- and 3',5'-linked phosphodiester bonds with and without incorporated C5'ppC groupings. An oligo(C) template containing exclusively 2',5'-phosphodiester bonds also serves as a template for the synthesis of complementary oligo(G)s. The oligo(C) template was prepared by the condensation of the 5'-phosphorimidazolide of cytidine on montmorillonite clay. These studies establish that RNA oligomers prepared by mineral catalysis, or other routes on the primitive earth, did not have to be exclusively 3',5'-linked to catalyze template-directed synthesis, since oligo(C)s containing a variety of linkage isomers serve as templates for the formation of complementary oligo(G)s. These findings support the postulate that origin of the RNA world was initiated by the RNA oligomers produced by polymerization of activated monomers formed by prebiotic processes.

Aluminum Silicates↗
PubMed1998

Structural and kinetic characterization of an acyl transferase ribozyme.

We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.

Acyltransferases↗
PubMed1998

Formation of oligonucleotide-PNA-chimeras by template-directed ligation.

DNA sequences have previously been reported to act as templates for the synthesis of PNA, and vice versa. A continuous evolutionary transition from an informational replicating system based on one polymer to a system based on the other would be facilitated if it were possible to form chimeras, that is molecules that contain monomers of both types. Here we show that ligation to form chimeras proceeds efficiently both on PNA and on DNA templates. The efficiency of ligation is primarily determined by the number of backbone bonds at the ligation site and the relative orientation of template and substrate strands. The most efficient reactions result in the formation of chimeras with ligation junctions resembling the structures of the backbones of PNA and DNA and with antiparallel alignment of both components of the chimera with the template, that is, ligations involving formation of 3'-phosphoramidate and 5'-ester bonds. However, double helices involving PNA are stable both with antiparallel and parallel orientation of the two strands. Ligation on PNA but not on DNA templates is, therefore, sometimes possible on templates with reversed orientation. The relevance of these findings to discussions of possible transitions between genetic systems is discussed.

DNA↗
PubMed1999

Efficient transfer of information from hexitol nucleic acids to RNA during nonenzymatic oligomerization.

Hexitol nucleic acids (HNAs) are DNA analogues that contain the standard nucleoside bases attached to a phosphorylated 1,5-anhydrohexitol backbone. We find that HNAs support efficient information transfer in nonensymatic template-directed reactions. HNA heterosequences appeared to be superior to the corresponding DNA heterosequences in facilitating synthesis of complementary oligonucleotides from nucleoside-5'-phosphoro-2-methyl imidazolides.

Chromatography, High Pressure Liquid↗
PubMed2004

Applying combinatorial chemistry and biology to food research.

In the past decade combinatorial chemistry has become a major focus of research activity in the pharmaceutical industry for accelerating the development of novel therapeutic compounds. The same combinatorial strategies could be applied to a broad spectrum of areas in agricultural and food research, including food safety and nutrition, development of product ingredients, and processing and conversion of natural products. In contrast to "rational design", the combinatorial approach relies on molecular diversity and high-throughput screening. The capability of exploring the structural and functional limits of a vast population of diverse chemical and biochemical molecules makes it possible to expedite the creation and isolation of compounds of desirable and useful properties. Several studies in recent years have demonstrated the utility of combinatorial methods for food research. These include the discovery of synthetic antimicrobial, antioxidative, and aflatoxin-binding peptides, the identification and analysis of unique flavor compounds, the generation of new enzyme inhibitors, the development of therapeutic antibodies for botulinum neurotoxins, the synthesis of unnatural polyketides and carotenoids, and the modification of food enzymes with novel properties. The results of such activities could open a large area of applications with potential benefits to the food industry. This review describes the current techniques of combinatorial chemistry and their applications, with emphasis on examples in food science research.

Combinatorial Chemistry Techniques↗
PubMed2002

Monocyclic human tachykinin NK-2 receptor antagonists as evolution of a potent bicyclic antagonist: QSAR and site-directed mutagenesis studies.

A new series of monocyclic pseudopeptidic tachykinin NK-2 receptor antagonists has been derived from nepadutant with the help of site-directed mutagenesis studies and QSAR models. MEN11558 is the lead compound which is evaluated on a series of 13 new human tachykinin NK-2 receptor mutants (Tyr107Ala, Gln109Ala, Asn110Ala, Phe112Ala, Ser164Phe, Cys167Gly, Phe168Ala, Tyr169Ala, Ile202Phe, Trp263Ala, Tyr269Phe, Tyr269Ala, and Phe293Ala) and 8 mutants on which data from nepadutant were already available (Gln166Ala, Ser170Ala, Thr171Ala, His198Ala, Tyr206Phe, Tyr266Phe, Tyr289Phe, and Tyr289Thr). The results show that the two compounds share most of their binding sites, in agreement with their hypothesized binding modes. This allows us to transfer the structural knowledge we already had for nepadutant to the new series of compounds. At the same time, a sound QSAR model is developed to assist the prioritization of new chemical syntheses. The result is the discovery of receptor antagonists with a higher affinity than nepadutant for the hNK-2 receptor.

Amino Acid Sequence↗