RESUMEN
The spatial distribution of cell surface proteins governs vital processes of the immune system such as intercellular communication and mobility. However, fluorescence microscopy has limited scalability in the multiplexing and throughput needed to drive spatial proteomics discoveries at subcellular level. We present Molecular Pixelation (MPX), an optics-free, DNA sequence-based method for spatial proteomics of single cells using antibody-oligonucleotide conjugates (AOCs) and DNA-based, nanometer-sized molecular pixels. The relative locations of AOCs are inferred by sequentially associating them into local neighborhoods using the sequence-unique DNA pixels, forming >1,000 spatially connected zones per cell in 3D. For each single cell, DNA-sequencing reads are computationally arranged into spatial proteomics networks for 76 proteins. By studying immune cell dynamics using spatial statistics on graph representations of the data, we identify known and new patterns of spatial organization of proteins on chemokine-stimulated T cells, highlighting the potential of MPX in defining cell states by the spatial arrangement of proteins.
Asunto(s)
Proteómica , Análisis de la Célula Individual , Proteómica/métodos , Análisis de la Célula Individual/métodos , Humanos , Linfocitos T/metabolismo , Análisis de Secuencia de ADN/métodosRESUMEN
The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells.
Asunto(s)
Biotecnología/métodos , Sistemas CRISPR-Cas , Edición Génica/métodos , Marcación de Gen/métodos , Línea Celular , ADN Helicasas/biosíntesis , ADN Helicasas/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Expresión Génica , Humanos , Células K562 , Oligonucleótidos/genética , ARN Guía de Kinetoplastida/metabolismo , Factor de Transcripción TFIIH/biosíntesis , Factor de Transcripción TFIIH/genética , TransfecciónRESUMEN
Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.
Asunto(s)
Sistemas CRISPR-Cas , ADN/genética , Edición Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Bases , Clonación Molecular , Citometría de Flujo , Expresión Génica , Marcación de Gen , Células HEK293 , Humanos , Microscopía Fluorescente , Modelos Moleculares , Plásmidos/genética , Conformación Proteica , ARN Guía de Kinetoplastida , Proteínas Recombinantes de Fusión/química , Relación Estructura-ActividadRESUMEN
The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.
Asunto(s)
Cromatina/química , Síndrome de Cockayne/genética , Histona Acetiltransferasas/genética , Histonas/metabolismo , Subunidades de Proteína/genética , Factor de Transcripción TFIIH/genética , Xerodermia Pigmentosa/genética , Acetilación , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Cromatina/metabolismo , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patología , Fibroblastos/citología , Fibroblastos/metabolismo , Edición Génica , Regulación de la Expresión Génica , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/metabolismo , Histonas/genética , Humanos , Modelos Biológicos , Osteoblastos/citología , Osteoblastos/metabolismo , Cultivo Primario de Células , Subunidades de Proteína/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Transcripción TFIIH/metabolismo , Iniciación de la Transcripción Genética , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patologíaRESUMEN
The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3'-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3'-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.
Asunto(s)
ADN/química , ADN/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Genes myc , Proteínas de Unión a Hierro/genética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , FrataxinaRESUMEN
Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
Asunto(s)
ADN Bacteriano/metabolismo , ADN Superhelicoidal/metabolismo , Glicina/análogos & derivados , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/antagonistas & inhibidores , ADN Bacteriano/química , ADN Superhelicoidal/química , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos/síntesis química , Oligonucleótidos Antisentido/síntesis química , Plásmidos/química , Plásmidos/metabolismo , Técnicas de Síntesis en Fase Sólida , Electricidad Estática , Relación Estructura-ActividadRESUMEN
In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.