Manipulation Of Biological Systems Research Articles

Synthetic Biology: From the Past to the Future

Synthetic biology is a highly promising interdisciplinary field aimed at integrating knowledge and techniques from various disciplines to achieve precise control and manipulation of biological systems. Since the discovery of DNA structure by Watson and Crick in 1953, synthetic biology has undergone rapid development. During this time, synthetic biologists have developed numerous tools and technologies to better understand biological systems and to develop new methods for constructing and manipulating these systems. Initially, synthetic biology research was mainly focused on genetic engineering, with the primary goal of inserting artificially synthesized DNA into biological systems to achieve specific functions. However, over time, this field has become increasingly complex, and researchers have begun exploring broader issues, such as how to integrate biological and non-biological systems and how to use these systems for medical and environmental applications. In recent years, synthetic biology research has reached new heights, involving the construction of artificially synthesized genomes and cells, and how to apply these systems to medical, energy, and environmental fields. These research achievements offer enormous potential for treating diseases, reducing pollution, and producing clean energy. In addition, the development of synthetic biology also involves artificial intelligence and machine learning, which can help accelerate the design and construction of biological systems, thus speeding up the progress of synthetic biology. Although synthetic biology still faces many challenges, the development prospects of this field are still very promising with the continuous advancement of technology. This article aims to provide an overview of the development of synthetic biology, including its historical background, early development, technological advances, applications, ethical and social impacts, and future directions, and explore the development process of synthetic biology over the past 60 years.

Integration of chemically modified nucleotides with DNA strand displacement reactions for applications in living systems.

Watson-Crick base pairing rules provide a powerful approach for engineering DNA-based nanodevices with programmable and predictable behaviors. In particular, DNA strand displacement reactions have enabled the development of an impressive repertoire of molecular devices with complex functionalities. By relying on DNA to function, dynamic strand displacement devices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation in living systems has been a slow process due to several persistent challenges, including nuclease degradation. To circumvent these issues, researchers are increasingly turning to chemically modified nucleotides as a means to increase device performance and reliability within harsh biological environments. In this review, we summarize recent progress toward the integration of chemically modified nucleotides with DNA strand displacement reactions, highlighting key successes in the development of robust systems and devices that operate in living cells and in vivo. We discuss the advantages and disadvantages of commonly employed modifications as they pertain to DNA strand displacement, as well as considerations that must be taken into account when applying modified oligonucleotide to living cells. Finally, we explore how chemically modified nucleotides fit into the broader goal of bringing dynamic DNA nanotechnology into the cell, and the challenges that remain. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing.

Systematically Studying the Effect of Small Molecules Interacting with RNA in Cellular and Preclinical Models.

The interrogation and manipulation of biological systems by small molecules is a powerful approach in chemical biology. Ideal compounds selectively engage a target and mediate a downstream phenotypic response. Although historically small molecule drug discovery has focused on proteins and enzymes, targeting RNA is an attractive therapeutic alternative, as many disease-causing or -associated RNAs have been identified through genome-wide association studies. As the field of RNA chemical biology emerges, the systematic evaluation of target validation and modulation of target-associated pathways is of paramount importance. In this Review, through an examination of case studies, we outline the experimental characterization, including methods and tools, to evaluate comprehensively the impact of small molecules that target RNA on cellular phenotype.

Kinetics of heterochiral strand displacement from PNA-DNA heteroduplexes.

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

Genetic Context Significantly Influences the Maintenance and Evolution of Degenerate Pathways.

Understanding the evolution of novel physiological traits is highly relevant for expanding the characterization and manipulation of biological systems. Acquisition of new traits can be achieved through horizontal gene transfer (HGT). Here, we investigate drivers that promote or deter the maintenance of HGT-driven degeneracy, occurring when processes accomplish identical functions through nonidentical components. Subsequent evolution can optimize newly acquired functions; for example, beneficial alleles identified in an engineered Methylorubrum extorquens strain allowed it to utilize a “Foreign” formaldehyde oxidation pathway substituted for its Native pathway for methylotrophic growth. We examined the fitness consequences of interactions between these alleles when they were combined with the Native pathway or both (Dual) pathways. Unlike the Foreign pathway context where they evolved, these alleles were often neutral or deleterious when moved into these alternative genetic backgrounds. However, there were instances where combinations of multiple alleles resulted in higher fitness outcomes than individual allelic substitutions could provide. Importantly, the genetic context accompanying these allelic substitutions significantly altered the fitness landscape, shifting local fitness peaks and restricting the set of accessible evolutionary trajectories. These findings highlight how genetic context can negatively impact the probability of maintaining native and HGT-introduced functions together, making it difficult for degeneracy to evolve. However, in cases where the cost of maintaining degeneracy was mitigated by adding evolved alleles impacting the function of these pathways, we observed rare opportunities for pathway coevolution to occur. Together, our results highlight the importance of genetic context and resulting epistasis in retaining or losing HGT-acquired degenerate functions.

Exploring Cryptococcus neoformans capsule structure and assembly with a hydroxylamine-armed fluorescent probe

Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.

Azobenzene-based small molecular photoswitches for protein modulation.

Molecular photoswitches are a class of chemical structures that can readily isomerize between distinct geometries upon irradiation with light. Molecular photoswitches are utilized to control protein structure and function with temporal and spatial precision. In this review, we summarize the recent progress in the development of azobenzene-based molecular photoswitches and their applications in the photocontrol of protein structure and function. For clarity of discussion, we divide the known photoswitchable proteins into different categories: protein motifs, ion channels, receptors, and enzymes. Basic approaches and considerations for the structure-guided design of photoswitchable ligands are discussed. The applications and limitations of current photoswitches are also discussed.

Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization.

Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.

Engineering intracellular biomineralization and biosensing by a magnetic protein.

Remote measurement and manipulation of biological systems can be achieved using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. Here we address this need by using high-throughput genetic screening in yeast to select variants of the iron storage ferritin (Ft) that display enhanced iron accumulation under physiological conditions. Expression of Ft mutants selected from a library of 107 variants induces threefold greater cellular iron loading than mammalian heavy chain Ft, over fivefold higher contrast in magnetic resonance imaging, and robust retention on magnetic separation columns. Mechanistic studies of mutant Ft proteins indicate that improved magnetism arises in part from increased iron oxide nucleation efficiency. Molecular-level iron loading in engineered Ft enables detection of individual particles inside cells and facilitates creation of Ft-based intracellular magnetic devices. We demonstrate construction of a magnetic sensor actuated by gene expression in yeast.

Optically controllable molecular logic circuits

Molecular logic circuits represent a promising technology for observation and manipulation of biological systems at the molecular level. However, the implementation of molecular logic circuits for temporal and programmable operation remains challenging. In this paper, we demonstrate an optically controllable logic circuit that uses fluorescence resonance energy transfer (FRET) for signaling. The FRET-based signaling process is modulated by both molecular and optical inputs. Based on the distance dependence of FRET, the FRET pathways required to execute molecular logic operations are formed on a DNA nanostructure as a circuit based on its molecular inputs. In addition, the FRET pathways on the DNA nanostructure are controlled optically, using photoswitching fluorescent molecules to instruct the execution of the desired operation and the related timings. The behavior of the circuit can thus be controlled using external optical signals. As an example, a molecular logic circuit capable of executing two different logic operations was studied. The circuit contains functional DNAs and a DNA scaffold to construct two FRET routes for executing Input 1 AND Input 2 and Input 1 AND NOT Input 3 operations on molecular inputs. The circuit produced the correct outputs with all possible combinations of the inputs by following the light signals. Moreover, the operation execution timings were controlled based on light irradiation and the circuit responded to time-dependent inputs. The experimental results demonstrate that the circuit changes the output for the required operations following the input of temporal light signals.

The Expanding Techniques of Progress: Agricultural Biotechnology and UN-REDD+

This paper provides a comparative analysis of agricultural biotechnology and the United Nations program for reduced emissions from deforestation and forest degradation (REDD). Despite the existing differences between the technical manipulation of biological systems and a conservation program aimed at reducing carbon and protecting forests, the two share commonalities in ideological origin, application, and values. Presented as positive developments, both seek to address large-scale issues such as global hunger and climate change, but while receiving national and international support they remain controversial issues. Both issues are critically assessed, beginning with a brief history, followed by the application of William Dugger's four invaluation processes: contamination, subordination, emulation, and mystification. This approach unravels the subtle social power of state and market forces that seek to control genetic material and forest frontiers as new outlets for growth and investment.

Tetracycline Determines the Conformation of Its Aptamer at Physiological Magnesium Concentrations

Synthetic riboswitches are versatile tools for the study and manipulation of biological systems. Yet, the underlying mechanisms governing its structural properties and regulation under physiological conditions are poorly studied. We performed spectroscopic and calorimetric experiments to explore the folding kinetics and thermodynamics of the tetracycline-binding aptamer, which can be employed as synthetic riboswitch, in the range of physiological magnesium concentrations. The dissociation constant of the ligand-aptamer complex was found to strongly depend on the magnesium concentration. At physiological magnesium concentrations, tetracycline induces a significant conformational shift from a compact, but heterogeneous intermediate state toward the completely formed set of tertiary interactions defining the regulation-competent structure. Thus, the switching functionality of the tetracycline-binding aptamer appears to include both a conformational rearrangement toward the regulation-competent structure and its thermodynamic stabilization.

Screening of Large Molecule Diversities by Phage Display

Molecules with tailored binding specificities are needed for many purposes such as the development of therapeutics, the detection and purification of biomolecules or in chemical biology for the study and manipulation of biological systems. With phage display technology, polypeptides with binding affinities to targets of interest can be isolated from billions of polypeptide variants with a modest amount of effort, time and cost. The technology was initially used for the generation and screening of peptide and antibody libraries and was later applied to many different protein scaffolds. More recently, chemically and structurally diverse molecule libraries were generated by chemically modifying phage-displayed polypeptides. In this article, the different classes of natural and non-natural structures that can be encoded and screened by phage display are reviewed with a special focus on bicyclic peptides that we routinely generate in our laboratory.

Arraying prostate specific antigen PSA and Fab anti‐PSA using light‐assisted molecular immobilization technology

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.

Chemical Biology Meets Biological Chemistry Minireview Series

Abstract What's the difference between and biology? One of us (JMG) received his Ph.D. at an institution where the biochemistry building was called the Laboratory of Biology, but that was a long time ago and things have changed. Biological chemistry (or biochemistry), that is the study of life processes at the molecular level, is now quite distinct from chemical biology. According to Wikipedia, Chemical is a scientific discipline spanning the fields of chemistry and that involves the application of chemical techniques and tools, often compounds produced through synthetic chemistry, to the study and manipulation of biological systems. Throughout the 105-year history of the Journal of Biological Chemistry, papers using techniques of chemistry to understand biological processes have appeared in the Journal, and in fact, many papers describing the development of chemical reagents and small molecule enzyme inhibitors top the all-time most-cited list of JBC papers (see http://www.jbc.org/reports/most-cited). However, several new journals have now emerged to emphasize the growing importance of biology as a unique discipline, albeit within the bounds of biological The following minireviews emphasize the importance of uniting synthetic chemistry with biochemistry toward understanding complex biological processes; in other words, how relevant biology is to chemistry. These reviews highlight both the application of chemical techniques toward understanding life processes at the molecular level (i.e., biochemistry) and the development of synthetic compounds either as tools for research or as candidate therapeutics for human diseases. These topics are naturally within the scope of interest of the JBC.

Direct Measurement of Heating by Optically Trapped Gold Nanoparticles Using Molecular Sorting in a Lipid Bilayer

Gold nanoparticles have are extremely useful as multi-functional and sensitive probes for investigation and manipulation of biological systems. Particles, as small as tenths of nanometers, can be visualized, optically manipulated, and used as controlled force transducers. Their high absorbance also makes them excellent converters of electromagnetic radiation into thermal energy. If the heating can be quantified it can also be advantageously used to perform controlled thermal treatment. We performed a direct measurement of the heating associated with optical trapping of individual gold nanospheres. A trapped gold nanosphere was embedded in a two dimensional supported gel phase lipid bilayer with incorporated fluorescent molecules which preferentially located, e.g., in the gel phase. Visualization of the melted region gave direct information about the temperature profile around the irradiated particle. The heating is highly dependent on particle size and laser power, with surface temperatures increasing from a few to hundreds of degrees Celcius. This quantification allows for creating controlled and localized temperature gradients which can be utilized for destruction of unwanted biological material such as cancer cells, to create local temperature gradients in lipid bilayers, or for nano-engineering purposes.

Ligand-directed tosyl chemistry for protein labeling in vivo

Here we describe a method for the site-selective attachment of synthetic molecules into specific 'endogenous' proteins in vivo using ligand-directed tosyl (LDT) chemistry. This approach was applied not only for chemically labeling proteins in living cells, tissues and mice but also for constructing a biosensor directly inside cells without genetic engineering. These data establish LDT chemistry as a new tool for the study and manipulation of biological systems.

Controlling cell behavior using their high-resolution light-sensing capability [Engineering in Genomics

To gain control over the cellular environment for the study and manipulation of biological systems, it is important to understand the signals and stimuli that provoke cellular mechanisms. Techniques for the perturbation and analysis at the single-cell resolution are crucial for understanding the behavior and mechanisms within the cellular environment. Thus, there is a need for the development of a new technology with the capability to manipulate and study individual cells. One promising technology utilizes light as a mechanism for delivering an active signal to cells. As light can be directed with exquisite precision in time and space, this presents an advantageous delivery system for initiating and controlling signaling events.

Chemical Biology: Past, Present and Future

Chemical biology (CB) is a scientific discipline spanning the fields of chemistry and biology involving the application of chemical techniques and tools, often natural products or small chemical compounds produced through synthetic chemistry, to the study and manipulation of biological systems. CB is providing new tools for deciphering protein modification and activity. Designer small molecules as probes of protein and cellular function are becoming the method of choice for careful profiling and development of biological processes in areas such as drug discovery, neurochemistry and molecular genetics. CB studies consists of three methodologies, chemical libraries with small molecules, high-throughput screening, and computational database. Combinatorial synthetic methods combined with high-throughput screening provided large numbers of potential lead compounds. CB reach is expanding into a vide range of fields and there is need to introduce CB to professional chemists working in biotechnology, pharmaceutical, agrochemical and other relevant sectors. Inorganic/organometallic/bioinorganic chemistry-chemical biology interface, biomolecular interactions (protein-protein, protein-carbohydrate), fluorescent quantum dots, biocongugation techniques, boronic acid-based chemosensors, click chemistry and artificial enzymes are described. CB aspects in aging, microfluidics, neurodegenerative diseases, human immunodeficiency virus as well as PROteolysis TArgeting Chimeric moleculeS (PROTACS) are reported. Research on the structural and energetic factors that distinguish specific protein interfaces should be strengthened. Exploration of protein networks should be actively pursued. Chemical biology new technologies may accelerate the discovery of still elusive cures to neurodegenerative diseases. Keywords: Chemical biology, chemical space, biomolecular interactions, bioinorganic chemistry

The Ethics of Deriving Gametes from ES Cells

The Ethics of Deriving Gametes from ES Cells