RESUMEN
Precision genome editing technologies have transformed modern biology. These technologies have arisen from the redirection of natural biological machinery, such as bacteriophage lambda proteins for recombineering and CRISPR nucleases for eliciting site-specific double-strand breaks. Less well-known is a widely distributed class of bacterial retroelements, retrons, that employ specialized reverse transcriptases to produce noncoding intracellular DNAs. Retrons' natural function and mechanism of genetic transmission have remained enigmatic. However, recent studies have harnessed their ability to produce DNA in situ for genome editing and evolution. This review describes retron biology and function in both natural and synthetic contexts. We also highlight areas that require further study to advance retron-based precision genome editing platforms.
Asunto(s)
Bacterias/genética , ADN Bacteriano/genética , Genes Bacterianos/genética , Ingeniería Genética/métodos , Retroelementos/genéticaRESUMEN
The sequence-specific hybridization and molecular recognition properties of DNA support the construction of stimulus-responsive hydrogels with precisely controlled crosslink geometry. Here we show that, as predicted by the conformational selection mechanism, the response kinetics of such a hydrogel can be tuned over orders of magnitude by modulating the thermodynamic stability of its crosslinks.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Hidrogeles/química , Cinética , Conformación MolecularRESUMEN
Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (â¼6 M) to low (â¼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.
Asunto(s)
Proteínas/química , Colorantes , Transferencia Resonante de Energía de Fluorescencia , Guanidina , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Molecular , Polietilenglicoles/química , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclusion of cumbersome washing and dilution steps or via careful control experimental conditions. Nature, in contrast, cannot afford the luxury of washing and dilution steps, nor can she arbitrarily change the conditions (temperature, pH, ionic strength) under which binding occurs in the homeostatically maintained environment within the cell. This forces evolution to focus at least as much effort on achieving optimal sensitivity and specificity as on achieving low detection limits, leading to the "invention" of a number of mechanisms, such as allostery and cooperativity, by which the useful dynamic range of receptors can be tuned, extended, narrowed, or otherwise optimized by design, rather than by sample manipulation. As the use of biomolecular receptors in artificial technologies matures (i.e., moves away from multistep, laboratory-bound processes and toward, for example, systems supporting continuous in vivo measurement) and these technologies begin to mimic the reagentless single-step convenience of naturally occurring chemoperception systems, the ability to artificially design receptors of enhanced sensitivity and specificity will likely also grow in importance. Thus motivated, we have begun to explore the adaptation of nature's solutions to these problems to the biomolecular receptors often employed in artificial biotechnologies. Using the population-shift mechanism, for example, we have generated nested sets of receptors and allosteric inhibitors that greatly expanded the normally limited (less than 100-fold) useful dynamic range of unmodified molecular and aptamer beacons, enabling the single-step (e.g., dilution-free) measurement of target concentrations across up to 6 orders of magnitude. Using this same approach to rationally introduce sequestration or cooperativity into these receptors, we have likewise narrowed their dynamic range to as little as 1.5-fold, vastly improving the sensitivity with which they respond to small changes in the concentration of their target ligands. Given the ease with which we have been able to introduce these mechanisms into a wide range of DNA-based receptors and the rapidity with which the field of biomolecular design is maturing, we are optimistic that the use of these and similar naturally occurring regulatory mechanisms will provide viable solutions to a range of increasingly important analytical problems.
Asunto(s)
Técnicas Biosensibles/métodos , ADN de Cadena Simple/química , Sondas Moleculares/química , Fenómenos Bioquímicos , ADN de Cadena Simple/genética , Límite de Detección , Sondas Moleculares/genética , MutaciónRESUMEN
Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and "smart" materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using "Hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.
Asunto(s)
Biotecnología/métodos , Ingeniería de Proteínas/métodos , Biología Sintética/métodos , Sitio Alostérico , Técnicas Biosensibles , Cocaína/química , ADN/química , Doxorrubicina/química , Fluoresceínas/química , Ligandos , Oxígeno/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , ARN Catalítico/química , Espectrometría de FluorescenciaRESUMEN
The high packing densities and fixed geometries with which biomolecules can be attached to macroscopic surfaces suggest that crowding effects may be particularly significant under these often densely packed conditions. Exploring this question experimentally, we report here the effects of crowding on the stability of a simple, surface-attached DNA stem-loop. We find that crowding by densely packed, folded biomolecules destabilizes our test-bed biomolecule by ~2 kJ/mol relative to the dilute (noninteracting) regime, an effect that presumably occurs due to steric and electrostatic repulsion arising from compact neighbors. Crowding by a dense brush of unfolded biomolecules, in contrast, enhances its stability by ~6 kJ/mol, presumably due to excluded volume and electrostatic effects that reduce the entropy of the unfolded state. Finally, crowding by like copies of the same biomolecule produces a significantly broader unfolding transition, likely because, under these circumstances, the stabilizing effects of crowding by unfolded molecules increase (and the destabilizing effects of neighboring folded molecules decrease) as more and more neighbors unfold. The crowding of surface-attached biomolecules may thus be a richer, more complex phenomenon than that seen in homogeneous solution.
Asunto(s)
Biopolímeros/química , ADN/química , Propiedades de Superficie , TermodinámicaRESUMEN
Allosteric cooperativity, which nature uses to improve the sensitivity with which biomolecular receptors respond to small changes in ligand concentration, could likewise be of use in improving the responsiveness of artificial biosystems. Thus motivated, we demonstrate here the rational design of cooperative molecular beacons, a widely employed DNA sensor, using a generalizable population-shift approach in which we engineer receptors that equilibrate between a low-affinity state and a high-affinity state exposing two binding sites. Doing so we achieve cooperativity within error of ideal behavior, greatly steepening the beacon's binding curve relative to that of the parent receptor. The ability to rationally engineer cooperativity should prove useful in applications such as biosensors, synthetic biology and "smart" biomaterials, in which improved responsiveness is of value.
Asunto(s)
Receptores de Droga/química , Sitios de Unión , Materiales Biocompatibles , Técnicas Biosensibles , ADN/química , Ligandos , Modelos Moleculares , Conformación Molecular , Ingeniería de Proteínas , Receptores de Droga/efectos de los fármacosRESUMEN
To ensure maximum specificity (i.e., minimize cross-reactivity with structurally similar analogues of the desired target), most bioassays invoke "stringency", the careful tuning of the conditions employed (e.g., pH, ionic strength, or temperature). Willingness to control assay conditions will fall, however, as quantitative, single-step biosensors begin to replace multistep analytical processes. This is especially true for sensors deployed in vivo, where the tuning of such parameters is not just inconvenient but impossible. In response, we describe here the rational adaptation of two strategies employed by nature to tune the affinity of biomolecular receptors so as to optimize the placement of their specificity "windows" without the need to alter measurement conditions: structure-switching and allosteric control. We quantitatively validate these approaches using two distinct, DNA-based receptors: a simple, linear-chain DNA suitable for detecting a complementary DNA strand and a structurally complex DNA aptamer used for the detection of a small-molecule drug. Using these models, we show that, without altering assay conditions, structure-switching and allostery can tune the concentration range over which a receptor achieves optimal specificity over orders of magnitude, thus optimally matching the specificity window with the range of target concentrations expected to be seen in a given application.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , ADNRESUMEN
The combination of modern biotechnologies such as DNA synthesis, λ red recombineering, CRISPR-based editing and next-generation high-throughput sequencing increasingly enables precise manipulation of genes and genomes. Beyond rational design, these technologies also enable the targeted, and potentially continuous, introduction of multiple mutations. While this might seem to be merely a return to natural selection, the ability to target evolution greatly reduces fitness burdens and focuses mutation and selection on those genes and traits that best contribute to a desired phenotype, ultimately throwing evolution into fast forward.
Asunto(s)
ADN/genética , Evolución Molecular Dirigida , Animales , Sistemas CRISPR-Cas , ADN/síntesis química , Ingeniería Genética , Genoma , MutaciónRESUMEN
The proteins of trans-acyltransferase modular polyketide synthases (PKSs) self-organize into assembly lines, enabling the multienzyme biosynthesis of complex organic molecules. Docking domains comprised of â¼25 residues at the C- and N-termini of these polypeptides (CDDs and NDDs) help drive this association through the formation of four-helix bundles. Molecular connectors like these are desired in synthetic contexts, such as artificial biocatalytic systems and biomaterials, to orthogonally join proteins. Here, the ability of six CDD/NDD pairs to link non-PKS proteins is examined using green fluorescent protein (GFP) variants. As observed through size-exclusion chromatography and Förster resonance energy transfer (FRET), matched but not mismatched pairs of Venus+CDD and NDD+mTurquoise2 fusion proteins associate with low micromolar affinities.
Asunto(s)
Simulación del Acoplamiento Molecular , Sintasas Poliquetidas/metabolismo , Secuencia de Aminoácidos , Cromatografía en Gel , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis , Péptidos/química , Péptidos/metabolismo , Sintasas Poliquetidas/químicaRESUMEN
Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have engineered completely synthetic, symmetrical oligomers by combining pairs of oppositely supercharged variants of a normally monomeric model protein through a strategy we term 'supercharged protein assembly' (SuPrA). We show that supercharged variants of green fluorescent protein can assemble into a variety of architectures including a well-defined symmetrical 16-mer structure that we solved using cryo-electron microscopy at 3.47 Å resolution. The 16-mer is composed of two stacked rings of octamers, in which the octamers contain supercharged proteins of alternating charges, and interactions within and between the rings are mediated by a variety of specific electrostatic contacts. The ready assembly of this structure suggests that combining oppositely supercharged pairs of protein variants may provide broad opportunities for generating novel architectures via otherwise unprogrammed interactions.
Asunto(s)
Multimerización de Proteína , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/metabolismo , Biología Sintética/métodos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Modelos Moleculares , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Electricidad EstáticaRESUMEN
While several genome editing methods exist, few are suitable for the continuous evolution of targeted sequences. Here we develop bacterial retroelements known as "retrons" for the dynamic, in vivo editing and mutagenesis of targeted genes. We first optimized retrons' ability to introduce preprogrammed mutations, optimizing both their expression and the host machinery that interacts with them to increase the incorporation frequency of mutations 78-fold over rates previously reported in synthetic systems. The optimized system is capable of simultaneously overwriting 13 separate positions spanning a 31-base length, and is for the first time shown to yield targeted deletions and insertions. To engineer retrons as a tool to introduce novel, unprogrammed mutations in specific targeted regions, we expressed them under a mutagenic T7 RNA polymerase. This coupled mutagenic T7 RNA polymerase-retron system enabled the evolution of diverse variants of environmentally selected antibiotic resistance genes, producing mutation rates in the targeted region 190-fold higher than background cellular mutation rates, potentially enabling the dynamic, continuous self-evolution of selected phenotypes.
Asunto(s)
Evolución Molecular Dirigida , Edición Génica/métodos , Retroelementos/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Mutagénesis , Regiones Promotoras Genéticas , Proteínas Virales/genéticaRESUMEN
Recent years have seen increasing study of stimulus-responsive hydrogels constructed from aptamer-connected DNA building blocks. Presumably due to a lack of simple, quantitative tools with which to measure gel responsiveness, however, the literature describing these materials is largely qualitative. In response, we demonstrate here simple, time-resolved, multiscale methods for measuring the response kinetics of these materials. Specifically, by employing trace amounts of fluorophore-quencher labeled cross-linkers and the rheology of entrapped fluorescent particles, we simultaneously measure dissolution at molecular, hundred-nanometer, and hundred-micron length-scales. For our test-bed system, an adenine-responsive hydrogel, we find biphasic response kinetics dependent on both effector concentration and depth within the gel and a dissolution pattern uniform at scales longer than a few times the monomer-monomer distance. Likewise, we find that, in agreement with theoretical predictions, dissolution kinetics over the hundred nanometer length scale exhibit a power-law-like dependence on the fraction of disrupted cross-links before a distinct crossover from solid-like to liquid-like behavior.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Hidrogeles/química , Cinética , Tamaño de la Partícula , Reología , Propiedades de SuperficieRESUMEN
Synthetically engineered organisms hold promise for a broad range of medical, environmental, and industrial applications. Organisms can potentially be designed, for example, for the inexpensive and environmentally benign synthesis of pharmaceuticals and industrial chemicals, for the cleanup of environmental pollutants, and potentially even for biomedical applications such as the targeting of specific diseases or tissues. However, the use of synthetically engineered organisms comes with several reasonable safety concerns, one of which is that the organisms or their genes could escape their intended habitats and cause environmental disruption. Here we review key recent developments in this emerging field of synthetic biocontainment and discuss further developments that might be necessary for the widespread use of synthetic organisms. Specifically, we discuss the history and modern development of three strategies for the containment of synthetic microbes: addiction to an exogenously supplied ligand; self-killing outside of a designated environment; and self-destroying encoded DNA circuitry outside of a designated environment.
RESUMEN
Recognition of the fundamental importance of allosteric regulation in biology dates back to not long after its discovery in the 1960s. Our ability to rationally engineer this potentially useful property into normally non-allosteric catalysts, however, remains limited. In response we report a DNA nanotechnology-enabled approach for introducing allostery into catalytic nucleic acids. Specifically, we have grafted one or two copies of a peroxidase-like DNAzyme, hemin-bound G-quadruplex (hemin-G), onto a DNA tetrahedral nanostructure in such a manner as to cause them to interact, modulating their catalytic activity. We achieve allosteric regulation of these catalysts by incorporating dynamically responsive oligonucleotides that respond to specific "effector" molecules (complementary oligonucleotides or small molecules), altering the spacing between the catalytic sites and thus regulating their activity. This designable approach thus enables subtle allosteric modulation in DNAzymes that is potentially of use for nanomedicine and nanomachines.
RESUMEN
Fluorescent, DNA-stabilized silver clusters are receiving much attention for sequence-selected colors and high quantum yields. However, limited knowledge of cluster structure is constraining further development of these "AgN-DNA" nanomaterials. We report the structurally sensitive, chiroptical activity of four pure AgN-DNA with wide ranging colors. Ubiquitous features in circular dichroism (CD) spectra include a positive dichroic peak overlying the lowest energy absorbance peak and highly anisotropic, negative dichroic peaks at energies well below DNA transitions. Quantum chemical calculations for bare chains of silver atoms with nonplanar curvature also exhibit these striking features, indicating electron flow along a chiral, filamentary metallic path as the origin for low-energy AgN-DNA transitions. Relative to the bare DNA, marked UV changes in CD spectra of AgN-DNA and silver cation-DNA solutions indicate that ionic silver content constrains nucleobase conformation. Changes in solvent composition alone can reorganize cluster structure, reconfiguring chiroptical properties and fluorescence.
Asunto(s)
ADN/química , Nanoestructuras/química , Plata/química , Transistores Electrónicos , Color , Modelos Moleculares , Conformación Molecular , Fenómenos Ópticos , Teoría Cuántica , Solventes/químicaRESUMEN
DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and many methylases have been regarded as promising targets for antimicrobial drugs. In this work, we report a stimulus responsive, self-regulating anticancer drug release platform, comprising a multifunctional DNA that upon methylation by methyltransferase (MTase) releases 5-fluorouracil (5-Fu) and in turn inhibits subsequent expression of MTase. The multifunctional DNA with anticancer drug are first methylated by DNA adenine methylation (DAM) methyltransferase (MTase) and then cut by the methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the functional DNA by the methylation/cleavage process will release the anticancer drug, resulting in inhibition of the activity of DAM in turn. Consequently, the enzyme activity of DAM MTase can be self-regulated. Furthermore, we found that the inhibition efficiency of 5-Fu significantly increase as it is functionalized with DNA.