Evolutionary biotechnology – theory,facts and perspectives

Molecular evolution has recently been applied in biotechnology which consist of the development of evolutionary strategies in the design of biopolymers with predefined properties and functions. At the heart of this new technology are the in vitro replication and random synthesis of RNA or DNA molecules, producing large libraries of genotypes that are subjected to selection techniques following DARWIN's principle. By means of these evolutionary methods, RNA molecules were derived which specifically bind to predefined target molecules. Ribozymes with new catalytic functions were obtained as well as RNA molecules that are resistant to cleavage by specific RNases. In addition, the catalytic specificities of group I introns, a special class of ribozymes, were modified by variation and selection. Efficient applications of molecular evolution to problems in biotechnology require a fundamental and detailed understanding of the evolutionary process. Two basic questions are of primary importance: (i) How can evolutionary methods be successful as the numbers of possible genotypes are so large that the chance of obtaining a particular sequence by random processes is practically zero, and (ii) how can populations avoid being caught in evolutionary traps corresponding to local fitness optima? This review is therefore concerned with an abridged account of the theory of molecular evolution, as well as its application to biotechnology. We add a brief discussion of new techniques for the massively parallel handling and screening of very small probes as is required for the spatial separation and selection of genotypes. Finally, some imminent prospects concerning the evolutionary design of biopolymers are presented.     

Hydroxyl radical footprinting of fluorescently labeled DNA

Footprinting is one of the simplest and most accurate approaches to investigate structure and interaction of biopolymers. It is based on the more difficult accessibility of intra- and intermolecular contacts for external damaging agents. According to this method, one end of polymer molecules is labeled before a sample is incubated with a damaging agent. The distribution of split products is used to conclude on the accessibility of different polymer regions under specific conditions. A variety of enzymatic and chemical splitting agents are used for footprinting. Currently, the highest temporal and spatial resolution without profound specificity to a nucleotide sequence can be reached with the use of hydroxyl radicals. A new variant of this approach, which suggests the use of DNA fluorescent labeling together with the present-day quantitative analysis, will allow extending the method’s boundaries.     

Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins

The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single‐molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high‐order molecular structures. For example, single‐stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two‐state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm‐like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two‐state transition between mechanically folded and unfolded states (such as proteins). © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1193–1199, 2014.     

Poly(3-mercaptopropionate): a nonbiodegradable biopolymer?

Polythioesters (PTEs) represent a novel class of biopolymers, which basically can be synthesized with polyhydroxyalkanoate (PHA) biosynthesis systems. Albeit technical applications of PTEs have not been elucidated yet, biodegradability might be an important property of this new thermoplastic material. In this study, extensive approaches were employed to isolate microorganisms capable of degrading poly(3-mercaptopropionate), poly(3MP), as a model compound of PTEs. Screening of 74 different environmental samples using various enrichment techniques were applied, but neither bacteria nor fungi could be isolated hydrolyzing poly(3MP). Furthermore, microcosms such as soil, compost, or activated sludge were applied to search for poly(3MP) degrading microorganisms, considering microbial communities and/or nonculturable bacteria, and the poly(3MP) material was exposed for more than half a year. However, no poly(3MP) degrading organisms were found, indicating an unexpected persistence of this biologically produced polymer.     

Comparative investigations of biopolymer hydration by physicochemical and modeling techniques.

The comparative investigation of biopolymer hydration by physicochemical techniques, particularly by small-angle X-ray scattering, has shown that the values obtained differ over a wide range, depending on the nature of the polymer and the environmental conditions. In the case of simple proteins, a large number of available data allow the derivation of a realistic average value for the hydration (0.35 g of water per gram of protein). As long as the average properties of proteins are considered, the use of such a default value is sufficient. Modeling approaches may be used advantageously, in order to differentiate between different assumptions and hydration contributions, and to correctly predict hydrodynamic properties of biopolymers on the basis of their three-dimensional structure. Problems of major concern are the positioning and the properties of the water molecules on the biopolymer surface. In this context, different approaches for calculating the molecular volume and surface of biopolymers have been applied, in addition to the development of appropriate hydration algorithms.     

Directed evolution of fungal laccases

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.     

Searching for protein signatures using a multilevel alphabet

Short motifs are known to play diverse roles in proteins, such as in mediating the interactions with other molecules, binding to membranes, or conducting a specific biological function. Standard approaches currently employed to detect short motifs in proteins search for enrichment of amino acid motifs considering mostly the sequence information. Here, we presented a new approach to search for common motifs (protein signatures) which share both physicochemical and structural properties, looking simultaneously at different features. Our method takes as an input an amino acid sequence and translates it to a new alphabet that reflects its intrinsic structural and chemical properties. Using the MEME search algorithm, we identified the proteins signatures within subsets of protein which encompass common sequence and structural information. We demonstrated that we can detect enriched structural motifs, such as the amphipathic helix, from large datasets of linear sequences, as well as predicting common structural properties (such as disorder, surface accessibility, or secondary structures) of known functional‐motifs. Finally, we applied the method to the yeast protein interactome and identified novel putative interacting motifs. We propose that our approach can be applied for de novo protein function prediction given either sequence or structural information. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Novel structural class of four disulfide-bridged peptides from Tityus serrulatus venom

A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.     

Oligonucleotide aptamers that recognize small molecules.

Nucleic acid receptors ('aptamers'), which recognize a large variety of organic molecules of low molecular weight, have been isolated from combinatorial nucleic acid libraries by in vitro selection methods. Structural studies of nucleic acid-small molecule complexes provide insight into both the principles of molecular recognition by this class of biopolymers and the architecture of tertiary motifs in nucleic acid folding. Aptamers that recognize small molecules are increasingly applied as tools in molecular biology, from the detection of oxidative damage in DNA to conditional gene expression and from their use as modules for the engineering of allosteric ribozymes to biosensors.     

Role of the biomolecular energy gap in protein design, structure, and evolution

The folding of natural biopolymers into unique three-dimensional structures that determine their function is remarkable considering the vast number of alternative states and requires a large gap in the energy of the functional state compared to the many alternatives. This Perspective explores the implications of this energy gap for computing the structures of naturally occurring biopolymers, designing proteins with new structures and functions, and optimally integrating experiment and computation in these endeavors. Possible parallels between the generation of functional molecules in computational design and natural evolution are highlighted.     

Development of ligase-assisted spacer addition for the measurement of microsatellites

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method.     

Acoustic communication in Drosophila

Acoustic communication during courtship has been extensively studied in many Drosophila species. Here we summarise approaches that have been applied to the study of both song production and hearing. These approaches harnessed a variety of genetic tools available in Drosophila, such as isolation of song or hearing mutants, QTL mapping and transgenesis as well as electrophysiology and behavioural analysis. We also provide a short guide for the methodology used in acoustic studies in Drosophila and discuss prospects and new tools that would benefit future research.     

Measuring the folding transition time of single RNA molecules

We describe a new, time-apertured photon correlation method for resolving the transition time between two states of RNA in folding--i.e., the time of the transition between states rather than the time spent in each state. Single molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy are used to obtain these measurements. Individual RNA molecules are labeled with fluorophores such as Cy3 and Cy5. Those molecules are then immobilized on a surface and observed for many seconds during which time the molecules spontaneously switch between two conformational states with different levels of flourescence resonance energy transfer efficiency. Single photons are counted from each fluorophore and cross correlated in a small window around a transition. The average of over 1000 cross correlations can be fit to a polynomial, which can determine transition times as short as the average photon emission interval. We applied the method to the P4-P6 domain of the Tetrahymena group I self-splicing intron to yield the folding transition time of 240 micros. The unfolding time is found to be too short to measure with this method.     

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.     

Hydrodynamic multibead modeling: problems, pitfalls and solutions. 3. Comparison of new approaches for improved predictions of translational properties

Modeling simple and complex biopolymers in solution requires the shapes of these molecules to be approximated by bead modeling procedures, primarily for the prediction of hydrodynamic and scattering quantities. Though several bead modeling strategies (strict, shell and filling models) and a variety of computer programs (preferably the HYDRO suite by the García de la Torre group) are available, several subtle questions remain to be answered, in particular concerning the appropriate volume correction for intrinsic viscosity computations. In this context, various versions of the HYDRO programs and different types of volume corrections, as well as the novel, alternative program ZENO of the Mansfield group, were applied to a plethora of thoroughly designed multibead models of spherical, ellipsoidal, cylindrical and prismatic shapes. A critical comparison of the results obtained reveals a variety of new aspects, useful for many future applications. Among these, application of our recently suggested “reduced volume correction” (RVC) together with specially adapted HYDRO versions and use of ZENO turned out to be highly effective, in particular when aiming at filling model strategies and using high bead numbers, a domain not fully supported by the recent HYDRO++ versions. By our approaches, the values of translational properties (diffusion coefficients, D, and intrinsic viscosities, [η]) of all multibead models applied were anticipated correctly.     

Mass measurement at high molecular weight--new tools for biotechnologists.

It has long been possible to analyse small molecules accurately and reproducibly by mass-spectrometric techniques. Two new techniques extend the application of mass spectrometry to proteins and other biopolymers of high molecular mass. The accuracy, sensitivity and resolving power of these new methods permits the detection of minor, but biologically significant protein modifications.     

Hydration of sodium hyaluronate, NMR study of an oriented sample

A sample of highly oriented Na-hyaluronate was prepared with a wet--spinning method and equilibrated to 75% relative humidity. Its proton NMR spectrum was recorded as a function of the angle theta between the hyaluronate helix axis and the external magnetic field. It appeared as a singlet with an angle-dependent line width expressed by delta v o + A(1-3cos2 theta)2. This reveals a residual intramolecular dipolar interaction of anisotropically rotating water molecules. The merging of the expected doublet into a single line is assumed to be caused by proton exchange between water molecules. The result indicates a hydration similar to that of many other fibrous biopolymers at comparable relative humidities.     

Modeling the free solution and gel electrophoresis of biopolymers: the bead array-effective medium model

Free solution and gel electrophoresis is an extremely useful tool in the separation of biopolymers. The complex nature of biopolymers, coupled with the usefulness of electrophoretic methods, has stimulated the development of theoretical modeling over the last 30 years. In this work, these developments are first reviewed with emphasis on Boundary Element and bead methodologies that enable the investigator to design realistic models of biopolymers. In the present work, the bead methodology is generalized to include the presence of a gel through the Effective Medium model. The biopolymer is represented as a bead array. A peptide, for example, made up of N amino acids is modeled as 2N beads. Duplex DNA is modeled as a discrete wormlike chain consisting of touching beads. The technical details of the method are placed in three Appendices. To illustrate the accuracy and effectiveness of the approach, two applications are considered. Model studies on both the free solution mobility of 73 peptides ranging in size from 2 to 42 amino acids, and the mobility of short duplex DNA in dilute agarose gels are discussed.     

In vitro selection of nucleic acids and proteins: What are we learning?

For almost a decade, in vitro selection experiments have been used to isolate novel nucleic acids, peptides and proteins according to their function. Selection experiments have altered our perception of molecular mimicry and catalysis, and they appear to be more facile than rational design at generating biopolymers with desired properties. New methods that have been developed improve the power of functional strategies in ways that nature has already discovered - by expanding library size and facilitating the recombination of positive mutations. Recent structural information on a number of selected and evolved molecules highlights future challenges for design via rational approaches.     

Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H

To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.