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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↗
PubMed2005

Diverse evolutionary trajectories characterize a community of RNA-cleaving deoxyribozymes: a case study into the population dynamics of in vitro selection.

Two parallel in vitro selections (denoted Selection A and Selection B) were conducted under different selection-pressure regimes, yielding a diverse community of RNA-cleaving deoxyribozymes. In Selection A, the reaction time was reduced four times (from 5 h to 5 s) over the course of 24 generations, while in Selection B the reaction time was maintained at 5 h for 30 rounds of selective amplification. Sequence alignment was conducted on more than 800 clones assembled from 18 generations that span both selections. Many prominent catalytic sequence classes, including some that extend across both selections, were identified and used to construct fitness landscapes depicting their rise and fall over time. The landscapes from both selections exhibit similar global trends despite differences in population dynamics. Some deoxyribozymes were predominant in the early rounds of selection but gave way to other species that dominated in the middle rounds. Ultimately, these middle classes disappeared from the landscape in favor of new and presumably more fit deoxyribozyme sequence classes. The shape of these landscapes alludes to the presence of many latent deoxyribozymes in the initial library, which can only be accessed by changes in the selection pressure and/or by adaptive mutations. Basic computer simulations provide theoretical corroboration of the experimentally observed pattern of staggered sequence-class transitions across the fitness landscapes. These simulations model the influence of one or more contributing factors, including catalytic rate, folding efficiency, PCR amplification efficiency, and random mutagenesis. This is the first study which thoroughly documents the topography of a deoxyribozyme fitness landscape over many generations of in vitro selection.

Base Sequence↗
PubMed2001

Viral escape from the neutralizing antibody response: the lymphocytic choriomeningitis virus model.

In addition to CD8+ cytotoxic T lymphocyte (CTL) responses, neutralizing antibodies contribute substantially to the long-term immune control of noncytopathic viruses, as demonstrated during infection with the lymphocytic choriomeningitis virus (LCMV). The high virus load during the initial phase of an infection and the ability of this RNA virus to spontaneously acquire mutations are important prerequisites for escaping an ongoing immune response. In this context, LCMV escape from the humoral response by single point mutations in neutralizing envelope protein determinants may occur, particularly during conditions of CTL deficiency, leading to virus persistence.

Animals↗
PubMed2004

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli.

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28 degrees C showed better production of torulene than 37 degrees C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2xYT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and beta-carotene, in E. coli. In contrast, glycerol-containing LB, 2xYT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1-3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.

Aerobiosis↗
PubMed2001

Did psychrophilic enzymes really win the challenge?

Organisms living in permanently cold environments, which actually represent the greatest proportion of our planet, display at low temperatures metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. They produce cold-evolved enzymes partially able to cope with the reduction in chemical reaction rates and the increased viscosity of the medium induced by low temperatures. In most cases, the adaptation is achieved through a reduction in the activation energy, leading to a high catalytic efficiency, which possibly originates from an increased flexibility of either a selected area of or the overall protein structure. This enhanced plasticity seems in return to be responsible for the weak thermal stability of cold enzymes. These particular properties render cold enzymes particularly useful in investigating the possible relationships existing between stability, flexibility, and specific activity and make them potentially unrivaled for numerous biotechnological tasks. In most cases, however, the adaptation appears to be far from being fully achieved.

Adaptation, Physiological↗
PubMed2006

Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium.

A non-recombinant mutant of Escherichia coli B, strain SZ194, was developed that produces over 1 M D-lactate from glucose (or sucrose) in 72 h using mineral salts medium supplemented with 1 mM: betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Yields approached the theoretical maximum with only trace amounts of co-products. Chiral purity of D-lactate was estimated to be 95%. Specific and volumetric productivities for SZ194 in mineral salts medium (pH 7.5) with betaine were equivalent to those in Luria broth.

Base Sequence↗
PubMed2006

Longevity differences among lines artificially selected for developmental time and wing length in Drosophila buzzatii.

We assessed the indirect response of longevity in lines selected for wing length (WL) and developmental time (DT). Longevity in selection lines was compared to laboratory control lines and the offspring of recently collected females. Wild flies (W lines), flies from lines selected for fast development (F lines), and for fast development and large wing length (L lines) outlived control laboratory lines (C lines) and lines selected for fast development and short wing (S lines). The decline in longevity in S lines is in line with the idea that body size and longevity are correlated and may be the result of the fixation of alleles at loci affecting pleiotropically the two traits under selection and longevity. In addition, inbreeding and artificial selection affected the correlation between wing length and longevity that occurs in natural populations of Drosophila buzzatii, suggesting that correlations between traits are not a perdurable feature in a population.

Aging↗
PubMed2005

Recent advances in GFP folding reporter and split-GFP solubility reporter technologies. Application to improving the folding and solubility of recalcitrant proteins from Mycobacterium tuberculosis.

We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691-695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102-107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.

Bacterial Proteins↗
PubMed2006

Evolutionary computation and multimodal search: a good combination to tackle molecular diversity in the field of peptide design.

The awesome degree of structural diversity accessible in peptide design has created a demand for computational resources that can evaluate a multitude of candidate structures. In our specific case, we translate the peptide design problem to an optimization problem, and use evolutionary computation (EC) in tandem with docking to carry out a combinatorial search. However, the use of EC in huge search spaces with different optima may pose certain drawbacks. For example, EC is prone to focus a search in the first good region found. This is a problem not only because of the undesirable and automatic rejection of potentially good search space regions, but also because the found solution may be extremely difficult to synthesize chemically or may even be a false docking positive. In order to avoid rejecting potentially good solutions and to maximize the molecular diversity of the search, we have implemented evolutionary multimodal search techniques, as well as the molecular diversity metric needed by the multimodal algorithms to measure differences between various regions of the search space.

Algorithms↗