Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “directed evolution”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,639 records · Page 91Linked to original sources
PubMed1996

Identification of cyclized calmodulin antagonists from a phage display random peptide library.

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.

Amino Acid Sequence↗
PubMed1996

Discovery of a herbicidal lead using polymer-bound activated esters in generating a combinatorial library of amides and esters.

A combinatorial library containing mixtures of amides and esters was prepared through solid-phase chemistry. The advantages of using solid-phase chemistry over solution-phase chemistry to prepare this library are discussed. The library was screened through a high-throughput whole organism herbicidal assay upon which a mixture containing amides was found to have herbicidal activity. Deconvolution of the mixture provided N-(3-benzoylphenyl)-3-(1,1-dimethylethyl)-1-methyl)-1 H-pyrazole-5-carboxamide as a herbicidal lead with broadleaf and narrowleaf pre-emergence herbicidal activity as low as 100 g/ha on some weed species. This study represents the first report of an agrochemical discovered using a combinatorial approach.

Amides↗
PubMed1996

Automating combinatorial chemistry: a primer on benchtop robotic systems.

Benchtop robotic systems are inexpensive, flexible automation tools with potential applications in a wide array of disciplines such as combinatorial chemistry, high-throughput screening, and genomics. We explain the basic components of a benchtop system and explore factors to consider when purchasing or customizing a robot, such as automation benefits, vendor selection, and current system limitations. Issues involving system specification, software design, and hardware customization are then discussed. Additionally, system optimization, validation, and support are detailed. Given a properly designed and implemented system, the combinatorial laboratory can markedly increase compound synthesis and purification.

Computers↗
PubMed1995

A single-bead decode strategy using electrospray ionization mass spectrometry and a new photolabile linker: 3-amino-3-(2-nitrophenyl)propionic acid.

A new linker that employs a photosensitive 3-amino-3-(2-nitrophenyl)propionyl functionality (ANP-resin) has been developed for the preparation of C-terminal carboxamides. A wide range of carboxamides were prepared and identified using the ANP-resin and electrospray ionization mass spectrometry. A single bead containing tripeptide Fmoc-Asp-Arg(Tos)-Val-NH2 was isolated, photocleaved and the peptide was characterized by tandem mass spectrometry, thereby verifying a library decode strategy that avoids complex tagging procedures.

Amino Acid Sequence↗
PubMed1995

Multiple simultaneous synthesis of phenolic libraries.

A series of analogous arrays of small, non-peptidyl, non-oligomeric compounds were synthesized on polystyrene resin. With the aid of a functionally differentiated phenolic scaffold, the batch preparation of unique benzamide and urea resins was accomplished, which were further derivatized in modified 96-well plates. An efficient cleavage reaction of the phenyl benzoate link enabled the isolation of more than 600 phenolic compounds in milligram quantities that were suitable for direct biological screening. The technology described herein represents a facile, economical approach to non-peptidyl chemical diversity.

Chemistry, Organic↗
PubMed1995

Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: identification of potent calcium-independent antigens.

The detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the 'FLAG' sequence DYKDDDDK-NH2, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52 x 10(6) and 4 x 10(12) different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif-DYK_K_(-), and were recognized by mAb M1 in the absence of calcium with 50- to 100-fold higher affinity than the FLAG octapeptide (IC50 = 273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.

Amino Acid Sequence↗
PubMed1995

Libraries of random-sequence polypeptides produced with high yield as carboxy-terminal fusions with ubiquitin.

Libraries of random-sequence polypeptides have been shown to be valuable sources of novel molecules possessing a variety of useful biologic-like activities, some of which may hold promise as potential vaccines and therapeutics. Previous random peptide expression systems were limited to low levels of peptide production and often to short sequences. Here we describe a series of libraries designed for increased polypeptide length. Cloned as carboxy-terminal extensions of ubiquitin, the fusions were produced in E. coli at high levels, and were purified to homogeneity. The majority of the extension proteins examined could be cleaved from ubiquitin by treatment with a ubiquitin-fusion hydrolase. The libraries described here are appropriate sources of novel polypeptides with desired binding or catalytic function, as well as tools with which to examine inherent properties of proteins as a whole. Toward the latter goal, we have examined structural properties of random-sequence proteins purified from these libraries. Quite surprisingly, fluorescence emission spectra of intrinsic tryptophan residues in several purified fusion proteins, under native-like and denaturing conditions, often resemble those expected for folded and unfolded states, respectively. The results presented here detail an important expansion in the range of potential uses for random-sequence polypeptide libraries.

Amino Acids↗
PubMed1995

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.

We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 x 10(9) members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of beta-endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v-ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.

Amino Acid Sequence↗
PubMed1995

Adaptive walks with noisy fitness measurements.

Adaptive walks constitute an optimization technique for searching a space of possible solutions, for example, a space of different molecules. The goal is to find a point in space (a molecule) that is optimal or near-optimal in some property, generally referred to as the 'fitness', such as its ability to bind to a given receptor. Adaptive walking, an analog of natural selection, is a powerful technique for searching landscapes. However, errors in the measurements will cause errors in the adaptive walks. Mutant molecules of higher fitness may be ignored or mutants of lower fitness may be accepted. To examine the effect of measurement error on adaptive walks, we simulate single-agent hill-climbing walks on NK landscapes of varying ruggedness where Gaussian noise is added to the fitness values to model measurement error. We consider both constant measurement noise and noise whose variance decays exponentially with fitness. We show that fitness-independent noise can cause walks to 'melt' off the peaks in a landscape, wandering in larger regions as the noise increases. However, we also show that a small amount of noise actually helps the walk perform better than with no noise. For walks in which noise decreases exponentially with fitness, the most characteristic behavior is that the walk meanders throughout the landscape until it stumbles across a point of relatively high fitness, then it climbs the landscape towards the nearest peak. Finally, we characterize the balance between selection pressure and noise and show that there are several classes of walk dynamic behavior.

Algorithms↗
PubMed1995

In vitro selection methodologies to probe RNA function and structure.

In vitro selection, or SELEX, has been used both to characterize the interaction of natural nucleic acids with proteins and to generate novel nucleic acid-binding species, or aptamers. Although numerous reports have demonstrated the power of the technique, they have not expanded on the methodologies that can be used for selection. This review focuses on the considerations and problems involved in selecting protein-binding aptamers from a random-sequence RNA pool. As an illustration, we describe two approaches to selecting aptamers to a particular target, the HTLV-I Rex protein. In the first, complete randomization is used to find an artificial, high-affinity RNA binding site. In the second, the contributions of individual nucleotides and/or base pairs to the natural Rex-binding element are determined by mutating the wild-type sequence and selecting active binding variants.

Base Sequence↗
PubMed1996

A new combination of protecting groups and links for encoded synthetic libraries suited for consecutive tests on the solid phase and in solution.

A strategy for high-throughput evaluation of combinatorial compound libraries is reported, which circumvents the necessity to test complex mixtures. The method is based on a new combination of protecting groups, solid-phase linker and tags. The bulk of the library first undergoes a binding assay with the components grafted on beads. A selection of beads carrying strong ligands is stripped from the labelled target and distributed into microvessels. The ligands are cleaved and rinsed into microeluates. Subsequently, a more detailed characterization with a functional assay in solution determines the best performers, which are identified through the peptidic tag left behind on the corresponding mother bead.

Chemistry, Organic↗
PubMed1996

Combinatorial synthesis of small-molecule libraries using 3-amino-5-hydroxybenzoic acid.

A non-peptide library of 2001 compounds has been prepared utilizing solid-phase techniques. The split/combine method was demonstrated to work well to form mixtures of compounds based on 3-amino-5-hydroxybenzoic acid as a core structure. The benzoic acid of the core structure served as the attachment point for the resin and the amino and hydroxy positions were variably substituted.

Aminobenzoates↗
PubMed1996

Solid phase organic synthesis (SPOS): a novel route to diketopiperazines and diketomorpholines.

The solid phase synthesis of libraries containing a 1,3,4,6-tetrasubstituted-2,5-diketo-1,4-piperazine scaffold (DKP) or a 3,4,6-trisubstituted-2,5-diketo-1,4-morpholine scaffold (DKM) from alpha-bromocarboxylic acids and amines is described. Using a design strategy which we refer to as divergent library design, both templates were prepared from a common intermediate. The general utility of this synthetic route in creating novel, non-peptidyl chemical libraries is discussed.

Chemistry, Organic↗
PubMed2003

Molecular adaptations to cold in psychrophilic enzymes.

Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earth's biosphere. To cope with the reduction of chemical reaction rates induced by low temperatures, these organisms synthesize enzymes characterized by a high catalytic activity at low temperatures associated, however, with low thermal stability. Thanks to recent advances provided by X-ray crystallography, protein engineering and biophysical studies, we are beginning to understand the molecular adaptations responsible for these properties which appear to be relatively diverse. The emerging picture suggests that psychrophilic enzymes utilize an improved flexibility of the structures involved in the catalytic cycle, whereas other protein regions if not implicated in catalysis may or may not be subjected to genetic drift.

Bacteria↗
PubMed2005

Parallel selections in vitro reveal a preference for 2'-5' RNA ligation upon deoxyribozyme-mediated opening of a 2',3'-cyclic phosphate.

We previously used in vitro selection to identify Mg(2+)-dependent deoxyribozymes that mediate the ligation reaction of an RNA 5'-hydroxyl group with a 2',3'-cyclic phosphate. In these efforts, all of the deoxyribozymes were identified via a common in vitro selection strategy, and all of the newly formed RNA linkages were non-native 2'-5' phosphodiester bonds rather than native 3'-5' linkages. Here we performed several new selections in which the relative arrangements of RNA and DNA were different as compared with the earlier studies. In all cases, we again find deoxyribozymes that create only 2'-5' linkages. This includes deoxyribozymes with an arrangement that favors 3'-5' linkages for a different chemical reaction, that of a 2',3'-diol plus 5'-triphosphate. These data indicate a strong and context-independent chemical preference for creating 2'-5' RNA linkages upon opening of a 2',3'-cyclic phosphate with a 5'-hydroxyl group. Preliminary assays show that some of the newly identified deoxyribozymes have promise for ligating RNA in a sequence-general fashion. Because 2',3'-cyclic phosphates are the products of uncatalyzed RNA backbone cleavage, their ligation reactions may be of direct relevance to the RNA World hypothesis.

Base Sequence↗
PubMed2005

Toward the selection of ribozymes for 1,3-dipolar cycloaddition reactions.

In vitro selection from combinatorial RNA libraries has repeatedly been used to study the catalytic and binding potential of nucleic acids. These selections not only led to RNA sequences catalyzing transformations known from metabolic pathways but also generated novel ribozymes for typical organic reactions. We were interested in 1,3-dipolar cycloaddition reactions, which are important tools for the formation of heterocyclic systems in organic chemistry and might also be found in the hypothetic RNA world. Here we describe our strategy and experiments to isolate RNA molecules catalyzing a 1,3-dipolar cycloaddition between nitrile oxides and an acrylate conjugated to RNA. We used direct selection with linker-coupled reactants, which has previously allowed the generation of true trans-acting catalysts for bimolecular reactions. A photocleavable linker was introduced to provide for a more stringent selection criterion. The 1,3-dipolar cycloaddition reaction was established in aqueous solution using a modified dinucleotide that was tethered to the dipolarophilic substrate. Two selection protocols were established, namely, a low-stringency affinity-based selection protocol, and a high-stringency procedure using the photocleavable moiety. In neither case was an increased activity toward the desired reaction obtained after 15 and 11 selection rounds, respectively. The resulting pools of RNA from several rounds were investigated both in cis and in trans. The limitations of this selection methodology are discussed in comparison with other catalysts for dipolar cycloadditions and, also, with respect to the unconventional substrates used.

Catalysis↗