RESUMEN
Electrochemical aptamer-based sensors support the high-frequency, real-time monitoring of molecules-of-interest in vivo. Achieving this requires methods for correcting the sensor drift seen during in vivo placements. While this correction ensures EAB sensor measurements remain accurate, as drift progresses it reduces the signal-to-noise ratio and precision. Here, we show that enzymatic cleavage of the sensor's target-recognizing DNA aptamer is a major source of this signal loss. To demonstrate this, we deployed a tobramycin-detecting EAB sensor analog fabricated with the DNase-resistant "xenonucleic acid" 2'O-methyl-RNA in a live rat. In contrast to the sensor employing the equivalent DNA aptamer, the 2'O-methyl-RNA aptamer sensor lost very little signal and had improved signal-to-noise. We further characterized the EAB sensor drift using unstructured DNA or 2'O-methyl-RNA oligonucleotides. While the two devices drift similarly in vitro in whole blood, the in vivo drift of the 2'O-methyl-RNA-employing device is less compared to the DNA-employing device. Studies of the electron transfer kinetics suggested that the greater drift of the latter sensor arises due to enzymatic DNA degradation. These findings, coupled with advances in the selection of aptamers employing XNA, suggest a means of improving EAB sensor stability when they are used to perform molecular monitoring in the living body.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Electroquímicas , Aptámeros de Nucleótidos/química , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , Animales , Ratas , Tobramicina/análisisRESUMEN
RNA function is increasingly appreciated to be more complex than merely communicating between DNA sequence and protein structure. RNA localization has emerged as a key contributor to the intricate roles RNA plays in the cell, and the link between dysregulated spatiotemporal localization and disease warrants an exploration beyond sequence and structure. However, the tools needed to visualize RNA with precise resolution are lacking in comparison to methods available for studying proteins. In the past decade, many techniques have been developed for imaging RNA, and in parallel super resolution and single-molecule techniques have enabled imaging of single molecules in cells. Of these methods, single molecule localization microscopy (SMLM) has shown significant promise for probing RNA localization. In this review, we highlight current approaches that allow super resolution imaging of specific RNA transcripts and summarize challenges and future opportunities for developing innovative RNA labeling methods that leverage the power of SMLM.
Asunto(s)
ARN , Imagen Individual de Molécula , Imagen Individual de Molécula/métodos , Microscopía Fluorescente/métodosRESUMEN
RNA editing or "epitranscriptomic modification" refers to the processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid. These chemical alterations can be found on either the ribose sugar or the nucleobase, and although many are "silent" and do not change the Watson-Crick-Franklin code of the RNA, others result in recoding events. More than 170 RNA modifications have been identified so far, each having a specific biological purpose. Additionally, dysregulated RNA editing has been linked to several types of diseases and disorders. As new modifications are discovered and our understanding of their functional impact grows, so does the need for selective methods of identifying and mapping editing sites in the transcriptome.The most common methods for studying RNA modifications rely on antibodies as affinity reagents; however, antibodies can be difficult to generate and often have undesirable off-target binding. More recently, selective chemical labeling has advanced the field by offering techniques that can be used for the detection, enrichment, and quantification of RNA modifications. In our method using acrylamide for inosine labeling, we demonstrated the versatility with which this approach enables pull-down or downstream functionalization with other tags or affinity handles. Although this method did enable the quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely because of the similar reactivity profiles of inosine and pseudouridine or other nucleobases.Seeking to overcome the inherent limitations of antibodies and chemical labeling methods, a more recent approach to studying the epitranscriptome is through the repurposing of proteins and enzymes that recognize modified RNA. Our laboratory has used Endonuclease V, a repair enzyme that cleaves inosine-containing RNAs, and reprogrammed it to instead bind inosine. We first harnessed EndoV to develop a preparative technique for RNA sequencing that we termed EndoVIPER-seq. This method uses EndoV to enrich inosine-edited RNAs, providing better coverage in RNA sequencing and leading to the discovery of previously undetected A-to-I editing sites. We also leveraged EndoV to create a plate-based immunoassay (EndoVLISA) to quantify inosine in cellular RNA. This approach can detect differential A-to-I editing levels across tissue types or disease states while being independent of RNA sequencing, making it cost-effective and high-throughput. By harnessing the molecular recognition capabilities of this enzyme, we show that EndoV can be repurposed as an "anti-inosine antibody" to develop new methods of detecting and enriching inosine from cellular RNA.Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications, and exploiting the affinity of these biomolecules offers a promising new direction for the field of epitranscriptomics.
Asunto(s)
Inosina , Edición de ARN , Inosina/química , Inosina/genética , Inosina/metabolismo , ARN/químicaRESUMEN
Enzyme activity requires sequential binding and chemical transformation of substrates. While directed evolution and random mutagenesis are common methods for improving catalytic activity, these methods do not allow for independent control of KM and kcat. To achieve such control, we envisioned that the colocalization of aptamers and enzymes that act on the same molecule could increase catalytic efficiency through preconcentration of substrate. We explored this concept with cocaine esterase and anticocaine aptamers having varying KD values, both encapsulated in MS2 virus-like particles. Rate enhancements were observed with magnitudes dependent on both aptamer:enzyme stoichiometry and aptamer KD, peaking when aptamer KD and enzyme KM were roughly equivalent. This beneficial effect was lost when either aptamer binding was too tight or the aptamers were not constrained to be close to the catalyst. This work demonstrates a modular way to enhance catalysis by independently controlling substrate capture and release to the processing enzyme.
Asunto(s)
Aptámeros de Nucleótidos , Catálisis , Aptámeros de Nucleótidos/química , CinéticaRESUMEN
Aptamers are widely used in small molecule detection applications due to their specificity, stability, and cost effectiveness. One key challenge in utilizing aptamers in sensors is matching the binding affinity of the aptamer to the desired concentration range for analyte detection. The most common methods for modulating affinity have inherent limitations, such as the likelihood of drastic changes in aptamer folding. Here, we propose that substituting guanosine for inosine at specific locations in the aptamer sequence provides a less perturbative approach to modulating affinity. Inosine is a naturally occurring nucleotide that results from hydrolytic deamination of adenosine, and like guanine, it base pairs with cytosine. Using the well-studied cocaine binding aptamer, we systematically replaced guanosine with inosine and were able to generate sequences having a range of binding affinities from 230 nM to 80 µM. Interestingly, we found that these substitutions could also modulate the specificity of the aptamers, leading to a range of binding affinities for structurally related analytes. Analysis of folding stability via melting temperature shows that, as expected, aptamer structure is impacted by guanosine-to-inosine substitutions. The ability to tune binding affinity and specificity through guanosine-to-inosine substitution provides a convenient and reliable approach for rapidly generating aptamers for diverse biosensing applications.
Asunto(s)
Aptámeros de Nucleótidos , Adenosina , Aptámeros de Nucleótidos/química , Guanosina , InosinaRESUMEN
Small-molecule toxins pose a significant threat to human health and the environment, and their removal is made challenging by their low molecular weight. Aptamers show promise as affinity reagents for binding these toxins, and recently, aptamers have been utilized for both sensing and remediation applications. We found that functionalization of ultrafiltration membranes with aptamers provides a convenient scaffold for toxin sequestration, but our initial efforts in this area were limited by low functionalization efficiencies and the ability to only capture a single target molecule. Herein, we describe detailed optimization of our aptamer-functionalized ultrafiltration membrane system and subsequent use for simultaneous removal of multiple small-molecule toxins. We examine multiple critical components involved in fabricating and functionalizing the membranes, including PEG polymer molecular weight for membrane fabrication, grafting conditions for pMAA attachment, and coupling reagents for aptamer functionalization. This screening enabled us to identify a set of unique conditions in which we were able to achieve high flux, near quantitative yield for DNA attachment, and effective overall depletion of both toxins and bacterial cells. Furthermore, we demonstrate the attachment of multiple aptamers and subsequent parallel removal of atrazine, bisphenol A, and microcystin-LR in a complex lake water matrix. Our rigorous evaluation resulted in depletion of multiple small-molecule toxins, contaminants, and microorganisms, demonstrating the potential of aptamer-functionalized membranes as point-of-use decontamination systems.
Asunto(s)
Aptámeros de Nucleótidos , Toxinas Marinas , Microcistinas , UltrafiltraciónRESUMEN
Adenosine-to-inosine (A-to-I) editing is a conserved eukaryotic RNA modification that contributes to development, immune response, and overall cellular function. Here, we utilize Endonucleaseâ V (EndoV), which binds specifically to inosine in RNA, to develop an EndoV-linked immunosorbency assay (EndoVLISA) as a rapid, plate-based chemiluminescent method for measuring global A-to-I editing signatures in cellular RNA. We first optimize and validate our assay with chemically synthesized oligonucleotides. We then demonstrate rapid detection of inosine content in treated cell lines, demonstrating equivalent performance against current standard RNA-seq approaches. Lastly, we deploy our EndoVLISA for profiling differential A-to-I RNA editing signatures in normal and diseased human tissue, illustrating the utility of our platform as a diagnostic bioassay. Together, the EndoVLISA method is cost-effective, straightforward, and utilizes common laboratory equipment, offering a highly accessible new approach for studying A-to-I editing. Moreover, the multi-well plate format makes this the first assay amenable for direct high-throughput quantification of A-to-I editing for applications in disease detection and drug development.
Asunto(s)
Adenosina/química , Inosina/química , Mediciones Luminiscentes , ARN/análisis , Humanos , Edición de ARNRESUMEN
Creating accurate maps of A-to-I RNA editing activity is vital to improving our understanding of the biological role of this process and harnessing it as a signal for disease diagnosis. Current RNA sequencing techniques are susceptible to random sampling limitations due to the complexity of the transcriptome and require large amounts of RNA material, specialized instrumentation, and high read counts to accurately interrogate A-to-I editing sites. To address these challenges, we show that Escherichia coli Endonuclease V (eEndoV), an inosine-cleaving enzyme, can be repurposed to bind and isolate A-to-I edited transcripts from cellular RNA. While Mg2+ enables eEndoV to catalyze RNA cleavage, we show that similar levels of Ca2+ instead promote binding of inosine without cleavage and thus enable high affinity capture of inosine in RNA. We leverage this capability to demonstrate EndoVIPER-seq (Endonuclease V inosine precipitation enrichment sequencing) as a facile and effective method to enrich A-to-I edited transcripts prior to RNA-seq, producing significant increases in the coverage and detection of identified editing sites. We envision the use of this approach as a straightforward and cost-effective strategy to improve the epitranscriptomic informational density of RNA samples, facilitating a deeper understanding of the functional roles of A-to-I editing.
Asunto(s)
Desoxirribonucleasa (Dímero de Pirimidina)/química , Inosina/química , Edición de ARN , ARN Mensajero/química , Calcio/química , Escherichia coli/enzimología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ARN/métodosRESUMEN
Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.
Asunto(s)
Glioxal/química , Ácidos Nucleicos/química , Secuencia de Bases , Catálisis , Dominio Catalítico , Metilación , Conformación de Ácido Nucleico , Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/química , Relación Estructura-Actividad , Biología Sintética , Tetrosas/química , TermodinámicaRESUMEN
Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Tirosina/análogos & derivados , Sitios de Unión , Colorantes Fluorescentes/química , Imagen Óptica , Tirosina/análisisRESUMEN
Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.
Asunto(s)
Acrilamida/química , Adenosina/análisis , Química Clic , Inosina/análisis , Edición de ARN , ARN/química , ARN/metabolismo , Adenosina/metabolismo , Inosina/metabolismoRESUMEN
Biomolecular assembly is a key driving force in nearly all life processes, providing structure, information storage, and communication within cells and at the whole organism level. These assembly processes rely on precise interactions between functional groups on nucleic acids, proteins, carbohydrates, and small molecules, and can be fine-tuned to span a range of time, length, and complexity scales. Recognizing the power of these motifs, researchers have sought to emulate and engineer biomolecular assemblies in the laboratory, with goals ranging from modulating cellular function to the creation of new polymeric materials. In most cases, engineering efforts are inspired or informed by understanding the structure and properties of naturally occurring assemblies, which has in turn fueled the development of predictive models that enable computational design of novel assemblies. This Review will focus on selected examples of protein assemblies, highlighting the story arc from initial discovery of an assembly, through initial engineering attempts, toward the ultimate goal of predictive design. The aim of this Review is to highlight areas where significant progress has been made, as well as to outline remaining challenges, as solving these challenges will be the key that unlocks the full power of biomolecules for advances in technology and medicine.
Asunto(s)
Péptidos/síntesis química , Polímeros/síntesis química , Proteínas/síntesis química , Modelos Moleculares , Péptidos/química , Polímeros/química , Proteínas/químicaRESUMEN
Despite advances in XNA evolution, the binding capabilities of artificial genetic polymers are currently limited to protein targets. Here, we describe the expansion of in vitro evolution techniques to enable selection of threose nucleic acid (TNA) aptamers to ochratoxin A (OTA). This research establishes the first example of an XNA aptamer of any kind to be evolved having affinity to a small-molecule target. Selection experiments against OTA yielded aptamers having affinities in the mid nanomolar range; with the best binders possessing KD values comparable to or better than those of the best previously reported DNA aptamer to OTA. Importantly, the TNA can be incubated in 50% human blood serum for seven days and retain binding to OTA with only a minor change in affinity, while the DNA aptamer is completely degraded and loses all capacity to bind the target. This not only establishes the remarkable biostability of the TNA aptamer, but also its high level of selectivity, as it is capable of binding OTA in a large background of competing biomolecules. Together, this research demonstrates that refining methods for in vitro evolution of XNA can enable the selection of aptamers to a broad range of increasingly challenging target molecules.
Asunto(s)
ADN/química , Ácidos Nucleicos/química , Ocratoxinas/aislamiento & purificación , Tetrosas/química , Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN/genética , Humanos , Ácidos Nucleicos/genética , Ocratoxinas/química , Técnica SELEX de Producción de Aptámeros , Tetrosas/genéticaRESUMEN
Nucleic acids and proteins are the fundamental biopolymers that support all life on Earth. Nucleic acids store large amounts of information in nucleobase sequences while peptides and proteins utilize diverse amino acid functional groups to adopt complex structures and perform wide-ranging activities. Although nature has evolved machinery to read the nucleic acid code and translate it into amino acid code, the extant biopolymers are restricted to encoding amino acid or nucleotide sequences separately, limiting their potential applications in medicine and biotechnology. Here we describe the design, synthesis, and stimuli-responsive assembly behavior of a bilingual biopolymer that integrates both amino acid and nucleobase sequences into a single peptide nucleic acid (PNA) scaffold to enable tunable storage and retrieval of tertiary structural behavior and programmable molecular recognition capabilities. Incorporation of a defined sequence of amino acid side-chains along the PNA backbone yields amphiphiles having a "protein code" that directs self-assembly into micellar architectures in aqueous conditions. However, these amphiphiles also carry a "nucleotide code" such that subsequent introduction of a complementary RNA strand induces a sequence-specific disruption of assemblies through hybridization. Together, these properties establish bilingual PNA as a powerful biopolymer that combines two information systems to harness structural responsiveness and sequence recognition. The PNA scaffold and our synthetic system are highly generalizable, enabling fabrication of a wide array of user-defined peptide and nucleotide sequence combinations for diverse future biomedical and nanotechnology applications.
Asunto(s)
Ácidos Nucleicos/genética , Ácidos Nucleicos de Péptidos/genética , Proteínas/genética , Secuencia de Bases , Código Genético , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Ácidos Nucleicos/síntesis química , Ácidos Nucleicos/química , Ácidos Nucleicos de Péptidos/síntesis química , Ácidos Nucleicos de Péptidos/química , Tensoactivos/síntesis química , Tensoactivos/químicaRESUMEN
The highly competitive nature of academic environments might seem to suggest that success can only be obtained at the cost of taking good care of oneself. However, sacrificing self-care can be extremely harmful. Herein, we explore ways that high performance and self-care can be mutually reinforcing and produce long-term success.
Asunto(s)
Rendimiento Académico/psicología , Autocuidado/métodos , Rendimiento Académico/normas , Éxito Académico , Humanos , Rendimiento Físico Funcional , Autocuidado/normasRESUMEN
Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive cross-linker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.
Asunto(s)
Colorantes Fluorescentes/química , Etiquetas de Fotoafinidad/química , Proteínas/química , Animales , Humanos , Microscopía Fluorescente , Colorantes de Rosanilina/químicaRESUMEN
Course-based undergraduate research experiences (CUREs) have gained traction as effective ways to expand the impact of undergraduate research while fulfilling pedagogical goals. In this Perspective, we present innovative ways to incorporate fundamental benefits and principles of CUREs into a classroom environment through information/technology-based research projects that lead to student-generated contributions to digital community resources (CoRes). These projects represent an attractive class of CUREs because they are less resource-intensive than laboratory-based CUREs, and the projects align with the expectations of today's students to create rapid and publicly accessible contributions to society. We provide a detailed discussion of two example types of CoRe projects that can be implemented in courses to impact research and education at the chemistry-biology interface: bioinformatics annotations and development of educational tools. Finally, we present current resources available for faculty interested in incorporating CUREs or CoRe projects into their pedagogical practices. In sharing these stories and resources, we hope to lower the barrier for widespread adoption of CURE and CoRe approaches and generate discussions about how to utilize the classroom experience to make a positive impact on our students and the future of the field of biochemistry.
Asunto(s)
Bioquímica/educación , Relaciones Comunidad-Institución , Biología Computacional , Colaboración Intersectorial , Investigación/organización & administración , Estudiantes , Humanos , InternetRESUMEN
Aptamers are short nucleic-acid biopolymers selected to have high affinity and specificity for protein or small-molecule target analytes. Aptamers can be engineered into split-aptamer biosensors comprising two nucleic acid strands that coassemble as they bind to a target, resulting in a large signal change from attached molecular probes (e.g., molecular beacons). The kinetics of split-aptamer assembly and their dependence on target recognition are largely unknown; knowledge of these kinetics could help in design and optimization of split-aptamer biosensors. In this work, we measure assembly kinetics of cocaine-dependent split-aptamer molecules using single-molecule fluorescence imaging. Assembly is monitored between a DNA strand tethered to a glass substrate and solutions containing the other strand tagged with a fluorescent label, with varying concentrations of the cocaine analyte. Dissociation rates are measured by tracking individual molecules and measuring their bound lifetimes. Dissociation-time distributions are biexponential, possibly indicating different folded states of the aptamer. The dissociation rate of only the longer-lived complex decreases with cocaine concentration, suggesting that cocaine stabilizes the long-lived aptamer complex. The variation in the slow dissociation rate with cocaine concentration is well described with an equilibrium-binding model, where the dissociation rate approaches a saturation limit consistent with the dissociation-equilibrium constant for cocaine-binding to the split aptamer. This single-molecule methodology provides a sensitive readout of cocaine-binding based on the dissociation kinetics of the split aptamer, allowing one to distinguish target-dependent aptamer assembly from background assembly. This methodology could be used to study other systems where target association affects the stability of aptamer duplexes.
Asunto(s)
Aptámeros de Nucleótidos/química , Cocaína/química , Sondas de ADN/química , Aptámeros de Nucleótidos/genética , Carbocianinas/química , Sondas de ADN/genética , Fluorescencia , Colorantes Fluorescentes/química , Cinética , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Imagen Individual de Molécula/métodosRESUMEN
Fluorescence-activated cell sorting (FACS) offers a powerful approach to high-throughput library screening in directed evolution experiments. However, FACS is rarely used in the evolution of stereoselective enzymes, due to the difficulty of designing fluorescence-based assays for measuring enantiopurity. Here, we describe a new FACS-based enantiopurity analysis approach that overcomes these limitations by using enantiomeric DNA biosensors labeled with orthogonal fluorophores. By co-encapsulating the biosensors with a mixture of target enantiomers in microfluidic droplets, we could demonstrate the use of FACS to differentiate between droplets having various levels of target enantiopurity. We envision the utility of this method for high-throughput screening of enantiopurity in the directed evolution of stereoselective enzymes, thereby facilitating the discovery of new asymmetric biocatalysts for the synthesis of pharmaceuticals and other high-value chemicals.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/química , Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Tirosina/análogos & derivados , Alcanosulfonatos/química , Compuestos Azo/química , Carbocianinas/química , Citometría de Flujo/instrumentación , Fluorescencia , Ensayos Analíticos de Alto Rendimiento/métodos , Dispositivos Laboratorio en un Chip , Estereoisomerismo , Tirosina/análisisRESUMEN
Adenosine-to-inosine (A-to-I) RNA editing is a widespread and conserved post-transcriptional modification, producing significant changes in cellular function and behavior. Accurately identifying, detecting, and quantifying these sites in the transcriptome is necessary to improve our understanding of editing dynamics, its broader biological roles, and connections with diseases. Chemical labeling of edited bases coupled with affinity enrichment has enabled improved characterization of several forms of RNA editing. However, there are no approaches currently available for pull-down of inosines. To address this need, we explore acrylamide as a labeling motif and report here an acrylamidofluorescein reagent that reacts with inosine and enables enrichment of inosine-containing RNA transcripts. This method provides improved sensitivity in the detection and identification of inosines toward a more comprehensive transcriptome-wide analysis of A-to-I editing. Acrylamide derivatization is also highly generalizable, providing potential for the labeling of inosine with a wide variety of probes and affinity handles.