RESUMEN
Electrochemical approaches, along with miniaturization of electrodes, are increasingly being employed to detect and quantify nucleic acid biomarkers. Miniaturization of the electrodes is achieved through the use of screen-printed electrodes (SPEs), which consist of one to a few dozen sets of electrodes, or by utilizing printed circuit boards. Electrode materials used in SPEs include glassy carbon (Chiang H-C, Wang Y, Zhang Q, Levon K, Biosensors (Basel) 9:2-11, 2019), platinum, carbon, and graphene (Cheng FF, He TT, Miao HT, Shi JJ, Jiang LP, Zhu JJ, ACS Appl Mater Interfaces 7:2979-2985, 2015). There are numerous modifications to the electrode surfaces as well (Cheng FF, He TT, Miao HT, Shi JJ, Jiang LP, Zhu JJ, ACS Appl Mater Interfaces 7:2979-2985, 2015). These approaches offer distinct advantages, primarily due to their demonstrated superior limit of detection without amplification. Using the SPEs and potentiostats, we can detect cells, proteins, DNA, and RNA concentrations in the nanomolar (nM) to attomolar (aM) range. The focus of this chapter is to describe the basic approach adopted for the use of SPEs for nucleic acid measurement.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Electrodos , Grafito , Grafito/química , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Ácidos Nucleicos/análisis , Humanos , ADN/análisisRESUMEN
Beyond its role as the bearer of genetic material, DNA also plays a crucial role in the activation phase of innate immunity. Pathogen recognition receptors (PRRs) and their homologs, pathogen-associated molecular patterns (PAMPs), form the foundation for driving innate immune activation and the induction of immune responses during infection. In the context of DNA viruses or bacterial infections, specific DNA sequences are recognized and bound by DNA sensors, marking the DNA as a PAMP for host recognition and subsequent activation of innate immunity. The primary DNA sensor pathway known to date is cGAS-STING, which can induce Type I interferons (IFN) and innate immune responses against viruses and bacteria. Additionally, the cGAS-STING pathway has been identified to mediate functions in autophagy and senescence. Herein, we introduce methods for using DNA PAMPs as molecular tools to study the role of cGAS-STING and its signaling pathway in regulating innate immunity, both in vitro and in vivo.
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ADN , Inmunidad Innata , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , ADN/metabolismo , ADN/genética , Animales , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , RatonesRESUMEN
The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed.
Asunto(s)
Cromosomas , Cromosomas/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , ADN/química , ADN/metabolismo , ADN/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Adenosina Trifosfato/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/químicaRESUMEN
Biomolecules contain various heterogeneities in their structures and local chemical properties, and their functions emerge through the dynamics encoded by these heterogeneities. Molecular dynamics model-based studies will greatly contribute to the elucidation of such chemical/mechanical structure-dynamics-function relationships and the mechanisms that generate them. Coarse-grained molecular dynamics models with appropriately designed nonuniform local interactions play an important role in considering the various phenomena caused by large molecular complexes consisting of various proteins and DNA such as nuclear chromosomes. Therefore, in this chapter, we will introduce a method for constructing a coarse-grained molecular dynamics model that simulates the global behavior of each chromosome in the nucleus of a mammalian cell containing many giant chromosomes.
Asunto(s)
Núcleo Celular , Simulación de Dinámica Molecular , Núcleo Celular/metabolismo , Núcleo Celular/química , Animales , Humanos , Cromosomas/química , ADN/química , ADN/metabolismo , MamíferosRESUMEN
Imaging-based spatial multi-omics technologies facilitate the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level, thereby enabling detailed observations of biological phenomena, including transcription machinery within cells and tissues. This chapter details the principles of such technologies, with a focus on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). A comprehensive step-by-step protocol for image analysis is provided, covering image preprocessing, spot detection, and data visualization. For practical application, complete Jupyter Notebook codes are made available on GitHub ( https://github.com/Ochiai-Lab/seqFISH_analysis ).
Asunto(s)
ADN , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , ARN , Programas Informáticos , Hibridación Fluorescente in Situ/métodos , ARN/genética , ARN/análisis , ARN/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , ADN/genética , Técnica del Anticuerpo Fluorescente/métodos , Humanos , AnimalesRESUMEN
While numerous methods exist for diagnosing tumors through the detection of miRNA within tumor cells, few can simultaneously achieve both tumor diagnosis and treatment. In this study, a novel graphene oxide (GO)-based DNA nanodevice (DND), initiated by miRNA, was developed for fluorescence signal amplification imaging and photodynamic therapy in tumor cells. After entering the cells, tumor-associated miRNA drives DND to Catalyzed hairpin self-assembly (CHA). The CHA reaction generated a multitude of DNA Y-type structures, resulting in a substantial amplification of Ce6 fluorescence release and the generation of numerous singlet oxygen (1O2) species induced by laser irradiation, consequently inducing cell apoptosis. In solution, DND exhibited high selectivity and sensitivity to miRNA-21, with a detection limit of 11.47 pM. Furthermore, DND discriminated between normal and tumor cells via fluorescence imaging and specifically generated O21 species in tumor cells upon laser irradiation, resulting in tumor cells apoptosis. The DND offer a new approach for the early diagnosis and timely treatment of malignant tumors.
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ADN , Grafito , MicroARNs , Fotoquimioterapia , Nanomedicina Teranóstica , Fotoquimioterapia/métodos , Humanos , MicroARNs/análisis , Grafito/química , Nanomedicina Teranóstica/métodos , ADN/química , Apoptosis/efectos de los fármacos , Imagen Óptica , Línea Celular Tumoral , Oxígeno Singlete/metabolismo , Oxígeno Singlete/química , Neoplasias/tratamiento farmacológico , Neoplasias/diagnóstico por imagenRESUMEN
Abstract Introduction: A recent revision of the generic classification of the Trochilidae based on DNA sequences revealed many inconsistencies with the current generic classification, largely based on plumage characters subject to homoplasy, especially in the Trochilini, the largest tribe. A thorough generic reorganization brought the classification into accord with the phylogeny, but due to lack of genetic data, two species remained unclassified. One of these was the Mangrove Hummingbird, "Amazilia" boucardi, endemic to Costa Rica and included in the IUCN red list of threatened species. Objective: To obtain molecular evidence to clarify the generic relationships of "A." boucardi. Methods: We isolated DNA from tissues of this species and amplified 4 nuclear and 4 mitochondrial fragments and compared these with homologous fragments from 56 species in the Trochilini, constructing phylogenetic trees with maximum likelihood and Bayesian methods. Results: Our phylogenetic analyses confirmed the placement of boucardi in the Trochilini and definitely excluded it from Amazilia but placed it with high confidence in the genus Chrysuronia Bonaparte, 1850, within which its closest relative is C. coeruleogularis, which also inhabits mangroves. Conclusions: Our genetic data based on nuclear and mitochondrial regions clearly indicate the relationship of A. boucardi and L. coeruleogularis. Moreover, it is also supported by their habitat distribution in the mangroves of the Pacific coast of Costa Rica and Western Panama. Therefore, we suggested to exclude A. boucardi as "incertae sedis".
Resumen Introducción: Una revisión reciente de la clasificación de la familia Trochilidae con base en secuencias de ADN demostró muchas incongruencias con la clasificación genérica previa, que había sido hecho con base en caracteres del plumaje muy sujetos a homoplasia, especialmente en la tribu más grande, Trochillini. Una reorganización de los géneros logró llevar su clasificación genérica a la concordancia con la filogenia, pero debido a la ausencia de datos genéticos, dos especies permanecieron sin clasificar. Una de estas fue el colibrí de manglar Amazilia boucardi, una especie endémica de Costa Rica, considerada como amenazada en la lista roja de la UICN. Objetivo: Obtener evidencia molecular para esclarecer las relaciones genéricas de A. boucardi. Métodos: Se aisló ADN de tejidos de esta especie y se amplificaron 4 fragmentos de ADN del núcleo y 5 de la mitocondria, y se compararon con fragmentos homólogos de 56 especies en la tribu Trochillini, generando árboles filogenéticos con métodos de máxima verosimilitud y bayesiano. Resultados: Los análisis filogénticos obtenidos confirmaron la ubicación de boucardi en Trochilini y definitivamente la excluyó del género Amazilia, pero la ubicó con un alto grado de confianza en el género Chrysuronia Bonaparte, 1850, dentro los cuales su pariente más cercano es C. coeruleogularis, que también habita manglares. Conclusiones: Nuestros datos genéticos basados en regiones nucleares y mitocondriales indican claramente la relación entre A. boucardi and L. coeruleogularis. Es más, lo anterior se sustenta por su distribución en los manglares de la costa Pacífica de Costa Rica y oeste de Panamá. Por lo tanto, sugerimos excluir a A. boucardi como "incertae sedis".
Asunto(s)
Animales , Aves/clasificación , ADN/análisis , Filogenia , Costa Rica , Genes MitocondrialesRESUMEN
Innovative chiral capillary silica monoliths (CSMs) were developed based on DNA nanoflowers (DNFs). Baseline separation of enantiomers such as atenolol, tyrosine, histidine, and nefopam was achieved by using DNF-modified CSMs, and the obtained resolution value was higher than 1.78. To further explore the effect of DNFs on enantioseparation, different types of chiral columns including DNA strand containing the complementary sequence of the template (DCT)-modified CSMs, DNF2-modified CSMs, and DNF3-modified CSMs were prepared as well. It was observed that DNF-modified CSMs displayed better chiral separation ability compared with DCT-based columns. The intra-day and inter-day repeatability of model analytes' retention time and resolution kept desirable relative standard deviation values of less than 8.28%. DNF2/DNF3-modified CSMs were able to achieve baseline separation of atenolol, propranolol, 2'-deoxyadenosine, and nefopam enantiomers. Molecular docking simulations were performed to investigate enantioselectivity mechanisms of DNA sequences for enantiomers. To indicate the successful construction of DNFs and DNF-modified CSMs, various charaterization approaches including scanning electron microscopy, agarose gel electrophoresis, dynamic light scattering analysis, electroosmotic flow, and Fourier-transform infrared spectroscopy were utilized. Moreover, the enantioseparation performance of DNF-modified CSMs was characterized in terms of sample volume, applied voltage, and buffer concentration. This work paves the way to applying DNF-based capillary electrochromatography microsystems for chiral separation.
Asunto(s)
ADN , Dióxido de Silicio , Dióxido de Silicio/química , ADN/química , ADN/aislamiento & purificación , Estereoisomerismo , Simulación del Acoplamiento Molecular , Atenolol/química , Atenolol/aislamiento & purificación , Nanoestructuras/química , Propranolol/química , Propranolol/aislamiento & purificaciónRESUMEN
The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.
Asunto(s)
Polietilenglicoles , Polietilenglicoles/química , Humanos , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/química , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Cloruro de Sodio/química , ADN/química , ADN/aislamiento & purificación , Polímeros/química , Biopsia Líquida/métodos , Sales (Química)/químicaRESUMEN
Telomeric G-quadruplexes (G4s) are non-canonical DNA structures composed of TTAGGG repeats. They are extensively studied both as biomolecules key for genome stability and as promising building blocks and functional elements in synthetic biology and nanotechnology. This is why it is extremely important to understand how the interaction between G4s is affected by their topology. We used small-angle x-ray scattering to investigate the end-to-end stacking of antiparallel telomeric G-quadruplexes formed by the sequence AG3(T2AG3)3. To represent the experimental data, we developed a highly efficient coarse-grained fitting tool, which successfully described the samples as an equilibrium mixture of monomeric and dimeric G4 species. Our findings indicate that the antiparallel topology prevents the formation of long multimeric structures under self-crowding conditions, unlike the hybrid/parallel structures formed by the same DNA sequence. This result supports the idea that the stacking of monomeric G-quadruplexes is strongly affected by the presence of diagonal loops.
Asunto(s)
G-Cuádruplex , Dispersión del Ángulo Pequeño , Telómero , Telómero/química , ADN/química , Difracción de Rayos XRESUMEN
There is an increasing need for determining 3D structures of DNAs, e.g., for increasing the efficiency of DNA aptamer selection. Recently, we have proposed a computational method of 3D structure prediction of DNAs, called 3dDNA, which has been integrated into our original web server 3dRNA, now renamed 3dRNA/DNA (http://biophy.hust.edu.cn/new/3dRNA). Currently, 3dDNA can only output the predicted DNA 3D structures for users but cannot rank them as an energy function for assessing DNA 3D structures is still lacking. Here, we first provide a brief introduction to 3dDNA and then introduce a new energy function, 3dDNAscore, for the assessment of DNA 3D structures. 3dDNAscore is an all-atom knowledge-based potential by integrating 86 atomic types from nucleic acids. Benchmarks demonstrate that 3dDNAscore can effectively identify near-native structures from the decoys generated by 3dDNA, thus enhancing the completeness of 3dDNA.
Asunto(s)
ADN , Modelos Moleculares , Conformación de Ácido Nucleico , ARN , ADN/química , ARN/química , Programas Informáticos , Biología Computacional/métodosRESUMEN
The ability to transform matter between numerous physical states or shapes without wires or external devices is a major challenge for robotics and materials design. Organisms can transform their shapes using biomolecules carrying specific information and localize at sites where transitions occur. Here, we introduce gel automata, which likewise can transform between a large number of prescribed shapes in response to a combinatorial library of biomolecular instructions. Gel automata are centimeter-scale materials consisting of multiple micro-segments. A library of DNA activator sequences can each reversibly grow or shrink different micro-segments by polymerizing or depolymerizing within them. We develop DNA activator designs that maximize the extent of growth and shrinking, and a photolithography process for precisely fabricating gel automata with elaborate segmentation patterns. Guided by simulations of shape change and neural networks that evaluate gel automata designs, we create gel automata that reversibly transform between multiple, wholly distinct shapes: four different letters and every even or every odd numeral. The sequential and repeated metamorphosis of gel automata demonstrates how soft materials and robots can be digitally programmed and reprogrammed with information-bearing chemical signals.
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Resinas Acrílicas , ADN , Geles , Geles/química , ADN/química , Resinas Acrílicas/química , Redes Neurales de la Computación , Algoritmos , Fenómenos Químicos , Secuencia de BasesRESUMEN
Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.
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Dominios Proteicos , Humanos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Evolución Molecular , ADN/química , ADN/metabolismo , Bases de Datos de Proteínas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Modelos Moleculares , Anotación de Secuencia Molecular , Bacterias/genética , Bacterias/metabolismoRESUMEN
Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52's ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.
Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Estructuras R-Loop , Proteína Recombinante y Reparadora de ADN Rad52 , Transcripción Genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Replicación del ADN/genética , Estructuras R-Loop/genética , Humanos , Daño del ADN , Roturas del ADN de Doble Cadena , ADN/metabolismo , ADN/genética , Reparación del ADN , Mutación , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
The challenge of transforming organized DNA structures into their metallized counterparts persists in the scientific field. In this context, utilizing DNA molecules modified with 7-deazapurine, provides a transformative solution. In this study, we present the solution structure of a DNA duplex that can be transformed into its metallized equivalent while retaining the natural base pairing arrangement through the creation of silver-modified Watson-Crick base pairs. Unlike previously documented X-ray structures, our research demonstrates the feasibility of preserving the intrinsic DNA self-assembly while incorporating AgI into the double helix, illustrating that the binding of silver does not disrupt the canonical base-pairing organization. Moreover, in our case, the uninterrupted AgI chain deviates from forming conventional straight linear chains; instead, it adheres to a helical arrangement dictated by the underlying DNA structure. This research challenges conventional assumptions and opens the door to precisely design structures based on the organization of highly stable Ag-DNA assemblies.
Asunto(s)
Emparejamiento Base , ADN , Conformación de Ácido Nucleico , Plata , Plata/química , ADN/química , Modelos Moleculares , SolucionesRESUMEN
This study improves the Logistic chaotic system and combines it with the hyperchaotic Chen system to create a dual chaotic system. The algorithm encrypts images in three stages. In the first stage, a plaintext-related key generation scheme is designed to generate the parameters and initial values of the dual chaotic system. In the second stage, the chaotic sequences generated by the dual chaotic system are used for dynamic DNA encoding and computation. In the third stage, the chaotic sequences generated by the improved Logistic chaotic system are used to perform row-column permutations, completing the scrambling. The security analysis of the encrypted images shows that the algorithm described in this paper is robust and secure, capable of resisting most known attacks. The algorithm is fast in encryption, provides high-quality image reconstruction, and is suitable for scenarios with high comprehensive performance and image quality requirements.
Asunto(s)
Algoritmos , Color , Seguridad Computacional , ADN , Procesamiento de Imagen Asistido por Computador , ADN/genética , Procesamiento de Imagen Asistido por Computador/métodos , Dinámicas no LinealesRESUMEN
Utilizing molecular dynamics and free energy perturbation, we examine the relative binding affinity of several covalent polycyclic aromatic hydrocarbon - DNA (PAH-DNA) adducts at the central adenine of NRAS codon-61, a mutational hotspot implicated in cancer risk. Several PAHs classified by the International Agency for Research on Cancer as probable, possible, or unclassifiable as to carcinogenicity are found to have greater binding affinity than the known carcinogen, benzo[a]pyrene (B[a]P). van der Waals interactions between the intercalated PAH and neighboring nucleobases, and minimal disruption of the DNA duplex drive increases in binding affinity. PAH-DNA adducts may be repaired by global genomic nucleotide excision repair (GG-NER), hence we also compute relative free energies of complexation of PAH-DNA adducts with RAD4-RAD23 (the yeast ortholog of human XPC-RAD23) which constitutes the recognition step in GG-NER. PAH-DNA adducts exhibiting the greatest DNA binding affinity also exhibit the least RAD4-RAD23 complexation affinity and are thus predicted to resist the GG-NER machinery, contributing to their genotoxic potential. In particular, the fjord region PAHs dibenzo[a,l]pyrene, benzo[g]chrysene, and benzo[c]phenanthrene are found to have greater binding affinity while having weaker RAD4-RAD23 complexation affinity than their respective bay region analogs B[a]P, chrysene, and phenanthrene. We also find that the bay region PAHs dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, and dibenzo[a,h]anthracene exhibit greater binding affinity and weaker RAD4-RAD23 complexation affinity than B[a]P. Thus, the study of PAH genotoxicity likely needs to be substantially broadened, with implications for public policy and the health sciences. This approach can be broadly applied to assess factors contributing to the genotoxicity of other unclassified compounds.
Asunto(s)
Aductos de ADN , Hidrocarburos Policíclicos Aromáticos , Hidrocarburos Policíclicos Aromáticos/toxicidad , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/metabolismo , Aductos de ADN/química , Aductos de ADN/metabolismo , Aductos de ADN/genética , Humanos , Reparación del ADN , Mutágenos/toxicidad , Mutágenos/química , Simulación de Dinámica Molecular , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Termodinámica , Benzo(a)pireno/toxicidad , Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , ADN/química , ADN/metabolismo , Benzopirenos/toxicidad , Benzopirenos/química , Benzopirenos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/químicaRESUMEN
Golden Gate assembly (GGA) can seamlessly generate full-length genes from DNA fragments. In principle, GGA could be used to design combinatorial mutation libraries for protein engineering, but creating accurate, complex, and cost-effective libraries has been challenging. We present GGAssembler, a graph-theoretical method for economical design of DNA fragments that assemble a combinatorial library that encodes any desired diversity. We used GGAssembler for one-pot in vitro assembly of camelid antibody libraries comprising >105 variants with DNA costs <0.007$ per variant and dropping significantly with increased library complexity. >93% of the desired variants were present in the assembly product and >99% were represented within the expected order of magnitude as verified by deep sequencing. The GGAssembler workflow is, therefore, an accurate approach for generating complex variant libraries that may drastically reduce costs and accelerate discovery and optimization of antibodies, enzymes and other proteins. The workflow is accessible through a Google Colab notebook at https://github.com/Fleishman-Lab/GGAssembler.
Asunto(s)
Mutación , Ingeniería de Proteínas/métodos , Ingeniería de Proteínas/economía , Biblioteca de Genes , ADN/genética , ADN/química , Biblioteca de PéptidosRESUMEN
Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.