RESUMO
The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.
Assuntos
Hibridização in Situ Fluorescente , Oligonucleotídeos , Hibridização in Situ Fluorescente/métodos , Humanos , Oligonucleotídeos/genética , Genômica/métodos , Análise de Célula Única/métodos , Corantes Fluorescentes/químicaRESUMO
Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50â¯mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44â¯% compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCDmax), allowing the determination of the minimum cell-specific perfusion rate (CSPRmin) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes.
Assuntos
Reatores Biológicos , Oligonucleotídeos , Rhodovulum , Oligonucleotídeos/genética , Rhodovulum/metabolismo , Rhodovulum/genética , Perfusão , Meios de Cultura/química , Técnicas de Cultura de Células/métodosRESUMO
Despite the unique ability of pioneer factors (PFs) to target nucleosomal sites in closed chromatin, they only bind a small fraction of their genomic motifs. The underlying mechanism of this selectivity is not well understood. Here, we design a high-throughput assay called chromatin immunoprecipitation with integrated synthetic oligonucleotides (ChIP-ISO) to systematically dissect sequence features affecting the binding specificity of a classic PF, FOXA1, in human A549 cells. Combining ChIP-ISO with in vitro and neural network analyses, we find that (1) FOXA1 binding is strongly affected by co-binding transcription factors (TFs) AP-1 and CEBPB; (2) FOXA1 and AP-1 show binding cooperativity in vitro; (3) FOXA1's binding is determined more by local sequences than chromatin context, including eu-/heterochromatin; and (4) AP-1 is partially responsible for differential binding of FOXA1 in different cell types. Our study presents a framework for elucidating genetic rules underlying PF binding specificity and reveals a mechanism for context-specific regulation of its binding.
Assuntos
Fator 3-alfa Nuclear de Hepatócito , Ligação Proteica , Fator de Transcrição AP-1 , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/genética , Sítios de Ligação , Células A549 , Cromatina/metabolismo , Cromatina/genética , Imunoprecipitação da Cromatina , Oligonucleotídeos/metabolismo , Oligonucleotídeos/genéticaRESUMO
Generating genetic diversity lies at the heart of directed evolution which has been widely used to engineer genetic parts and gene circuits in synthetic biology. With the ever-expanding application of directed evolution, different approaches of generating genetic diversity are required to enrich the traditional toolbox. Here we show in vitro generation of genetic diversity for directed evolution by error-prone artificial DNA synthesis (epADS). This approach comprises a three-step process which incorporates base errors randomly generated during chemical synthesis of oligonucleotides under specific conditions into the target DNA. Through this method, 200 ~ 4000 folds of diversification in fluorescent strength have been achieved in genes encoding fluorescent proteins. EpADS has also been successfully used to diversify regulatory genetic parts, synthetic gene circuits and even increase microbial tolerance to carbenicillin in a short time period. EpADS would be an alternative tool for directed evolution which may have useful applications in synthetic biology.
Assuntos
DNA , Evolução Molecular Direcionada , Variação Genética , Evolução Molecular Direcionada/métodos , DNA/genética , Biologia Sintética/métodos , Oligonucleotídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5'- and 3'-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.
Assuntos
SARS-CoV-2 , Software , SARS-CoV-2/genética , Reação em Cadeia da Polimerase/métodos , Oligonucleotídeos/química , Oligonucleotídeos/genética , COVID-19/virologia , Biologia Computacional/métodos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/químicaRESUMO
Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for in vivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially in vivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and in vivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening in vivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Oligonucleotídeos/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Técnica de Seleção de Aptâmeros/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Protein variant libraries produced by site-directed mutagenesis are a useful tool utilized by protein engineers to explore variants with potentially improved properties, such as activity and stability. These libraries are commonly built by selecting residue positions and alternative beneficial mutations for each position. All possible combinations are then constructed and screened, by incorporating degenerate codons at mutation sites. These degenerate codons often encode additional unwanted amino acids or even STOP codons. Our study aims to take advantage of annealing based recombination of oligonucleotides during synthesis and utilize multiple degenerate codons per mutation site to produce targeted protein libraries devoid of unwanted variants. Toward this goal we created an algorithm to calculate the minimum number of degenerate codons necessary to specify any given amino acid set, and a dynamic programming method that uses this algorithm to optimally partition a DNA target sequence with degeneracies into overlapping oligonucleotides, such that the total cost of synthesis of the target mutant protein library is minimized. Computational experiments show that, for a modest increase in DNA synthesis costs, beneficial variant yields in produced mutant libraries are increased by orders of magnitude, an effect particularly pronounced in large combinatorial libraries.
Assuntos
Mutação , Algoritmos , Proteínas/genética , Proteínas/química , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , DNA/genética , DNA/química , Oligonucleotídeos/química , Oligonucleotídeos/genéticaRESUMO
Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFß pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.
Assuntos
Processamento Alternativo , Inteligência Artificial , Aprendizado de Máquina , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Oligonucleotídeos Antissenso/genética , Movimento Celular/genética , Spliceossomos/metabolismo , Spliceossomos/genética , Oligonucleotídeos/genética , FemininoRESUMO
Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichia coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.
Assuntos
Escherichia coli , Oligonucleotídeos , Plasmídeos , Escherichia coli/genética , Oligonucleotídeos/genética , Plasmídeos/genética , Integrases/genética , Integrases/metabolismo , Genoma Bacteriano/genética , Engenharia Genética/métodos , Técnicas de Inativação de Genes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) that share identical or near-identical sequences constitute miRNA families and are predicted to act redundantly. Yet recent evidence suggests that members of the same miRNA family with high sequence similarity might have different roles and that this functional divergence might be rooted in their precursors' sequence. Current knock-down strategies such as antisense oligonucleotides (ASOs) or miRNA sponges cannot distinguish between identical or near identical miRNAs originating from different precursors to allow exploring unique functions of these miRNAs. We here develop a novel strategy based on short 2'-OMe/LNA-modified oligonucleotides to selectively target specific precursor molecules and ablate the production of individual members of miRNA families in vitro and in vivo. Leveraging the highly conserved Xenopus miR-181a family as proof-of-concept, we demonstrate that 2'-OMe/LNA-ASOs targeting the apical region of pre-miRNAs achieve precursor-selective inhibition of mature miRNA-5p production. Furthermore, we extend the applicability of our approach to the human miR-16 family, illustrating its universality in targeting precursors generating identical miRNAs. Overall, our strategy enables efficient manipulation of miRNA expression, offering a powerful tool to dissect the functions of identical or highly similar miRNAs derived from different precursors within miRNA families.
Assuntos
MicroRNAs , Oligonucleotídeos , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/química , Xenopus/genéticaRESUMO
Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.
Assuntos
DNA Nucleotidilexotransferase , Biologia Sintética , DNA Nucleotidilexotransferase/genética , Biologia Sintética/métodos , DNA/genética , DNA Polimerase Dirigida por DNA , Nucleotídeos/genética , Oligonucleotídeos/genéticaRESUMO
CRISPR-Cas systems with dual functions offer precise sequence-based recognition and efficient catalytic cleavage of nucleic acids, making them highly promising in biosensing and diagnostic technologies. However, current methods encounter challenges of complexity, low turnover efficiency, and the necessity for sophisticated probe design. To better integrate the dual functions of Cas proteins, we proposed a novel approach called CRISPR-Cas Autocatalysis Amplification driven by LNA-modified Split Activators (CALSA) for the highly efficient detection of single-stranded DNA (ssDNA) and genomic DNA. By introducing split ssDNA activators and the site-directed trans-cleavage mediated by LNA modifications, an autocatalysis-driven positive feedback loop of nucleic acids based on the LbCas12a system was constructed. Consequently, CALSA enabled one-pot and real-time detection of genomic DNA and cell-free DNA (cfDNA) from different tumor cell lines. Notably, CALSA achieved high sensitivity, single-base specificity, and remarkably short reaction times. Due to the high programmability of nucleic acid circuits, these results highlighted the immense potential of CALSA as a powerful tool for cascade signal amplification. Moreover, the sensitivity and specificity further emphasized the value of CALSA in biosensing and diagnostics, opening avenues for future clinical applications.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , DNA de Cadeia Simples , Oligonucleotídeos , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA/química , DNA/genética , Linhagem Celular Tumoral , CatáliseRESUMO
Insects are a highly diverse phylogeny and possess a wide variety of traits, including the presence or absence of wings and metamorphosis. These diverse traits are of great interest for studying genome evolution, and numerous comparative genomic studies have examined a wide phylogenetic range of insects. Here, we analyzed 22 insects belonging to a wide phylogenetic range (Endopterygota, Paraneoptera, Polyneoptera, Palaeoptera, and other insects) by using a batch-learning self-organizing map (BLSOM) for oligonucleotide compositions in their genomic fragments (100-kb or 1-Mb sequences), which is an unsupervised machine learning algorithm that can extract species-specific characteristics of the oligonucleotide compositions (genome signatures). The genome signature is of particular interest in terms of the mechanisms and biological significance that have caused the species-specific difference, and can be used as a powerful search needle to explore the various roles of genome sequences other than protein coding, and can be used to unveil mysteries hidden in the genome sequence. Since BLSOM is an unsupervised clustering method, the clustering of sequences was performed based on the oligonucleotide composition alone, without providing information about the species from which each fragment sequence was derived. Therefore, not only the interspecies separation, but also the intraspecies separation can be achieved. Here, we have revealed the specific genomic regions with oligonucleotide compositions distinct from the usual sequences of each insect genome, e.g., Mb-level structures found for a grasshopper Schistocerca americana. One aim of this study was to compare the genome characteristics of insects with those of vertebrates, especially humans, which are phylogenetically distant from insects. Recently, humans seem to be the "model organism" for which a large amount of information has been accumulated using a variety of cutting-edge and high-throughput technologies. Therefore, it is reasonable to use the abundant information from humans to study insect lineages. The specific regions of Mb length with distinct oligonucleotide compositions have also been previously observed in the human genome. These regions were enriched by transcription factor binding motifs (TFBSs) and hypothesized to be involved in the three-dimensional arrangement of chromosomal DNA in interphase nuclei. The present study characterized the species-specific oligonucleotide compositions (i.e., genome signatures) in insect genomes and identified specific genomic regions with distinct oligonucleotide compositions.
Assuntos
Genoma Humano , Genoma de Inseto , Animais , Humanos , Filogenia , Genoma de Inseto/genética , Oligonucleotídeos/genética , Inteligência ArtificialRESUMO
Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12 bp DNA or RNA duplex on its association (kon) and dissociation (koff) kinetics. We find that such defects have relatively small effects on the association kinetics, while the dissociation kinetics vary in a position-dependent manner by up to 6 orders of magnitude. Building upon this observation, we explored a competition scenario involving multiple oligonucleotides, and observed a transient low specificity of probe hybridization to fully versus partially complementary targets in solution. We characterize these long-lived metastable states and their evolution toward equilibrium, and show that sufficiently long-lived mis-paired duplexes can serve as substrates for prebiotically relevant chemical copying reactions. Our results suggest that transient low accuracy states may spontaneously emerge within all complex nucleic acid systems comprising a large enough number of competing strands, with potential repercussions for gene regulation in the realm of modern biology and the prebiotic preservation of genetic information.
Assuntos
Hibridização de Ácido Nucleico , Oligonucleotídeos , DNA/química , Cinética , Oligonucleotídeos/genética , Oligonucleotídeos/química , RNA/química , TermodinâmicaRESUMO
Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.
Assuntos
DNA , Sequências Repetitivas de Ácido Nucleico , Humanos , Hibridização in Situ Fluorescente/métodos , DNA/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sondas de Oligonucleotídeos/genética , Sondas de DNA/genética , Oligonucleotídeos/genéticaRESUMO
RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.
Assuntos
RNA Ligase (ATP) , RNA Ligase (ATP)/metabolismo , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/química , Especificidade por Substrato , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Planococáceas/enzimologia , Planococáceas/genética , Engenharia de Proteínas , Mutação , Modelos Moleculares , Trifosfato de Adenosina/metabolismo , Oligonucleotídeos/metabolismo , Oligonucleotídeos/genéticaRESUMO
For peanut, the lack of stable cytological markers is a barrier to tracking specific chromosomes, elucidating the genetic relationships between genomes and identifying chromosomal variations. Chromosome mapping using single-copy oligonucleotide (oligo) probe libraries has unique advantages for identifying homologous chromosomes and chromosomal rearrangements. In this study, we developed two whole-chromosome single-copy oligo probe libraries, LS-7A and LS-8A, based on the reference genome sequences of chromosomes 7A and 8A of Arachis duranensis. Fluorescence in situ hybridization (FISH) analysis confirmed that the libraries could specifically paint chromosomes 7 and 8. In addition, sequential FISH and electronic localization of LS-7A and LS-8A in A. duranensis (AA) and A. ipaensis (BB) showed that chromosomes 7A and 8A contained translocations and inversions relative to chromosomes 7B and 8B. Analysis of the chromosomes of wild Arachis species using LS-8A confirmed that this library could accurately and effectively identify A genome species. Finally, LS-7A and LS-8A were used to paint the chromosomes of interspecific hybrids and their progenies, which verified the authenticity of the interspecific hybrids and identified a disomic addition line. This study provides a model for developing specific oligo probes to identify the structural variations of other chromosomes in Arachis and demonstrates the practical utility of LS-7A and LS-8A.
Assuntos
Arachis , Coloração Cromossômica , Cromossomos de Plantas , Hibridização in Situ Fluorescente , Coloração Cromossômica/métodos , Cromossomos de Plantas/genética , Arachis/genética , Mapeamento Cromossômico , Oligonucleotídeos/genética , Translocação GenéticaRESUMO
INTRODUCTION: Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described. AREAS COVERED: Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results. EXPERT OPINION: Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.
Assuntos
Anticorpos , Oligonucleotídeos , Humanos , Oligonucleotídeos/genéticaRESUMO
Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Nucleotídeos , Análise de Sequência de RNA , Oligonucleotídeos/genética , Reação em Cadeia da PolimeraseRESUMO
Drugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce mechanistically interpretable quantitative models for the sequence-specific and concentration-dependent behavior of splice-modifying drugs. Using massively parallel splicing assays, RNA-seq experiments, and precision dose-response curves, we obtain quantitative models for two small-molecule drugs, risdiplam and branaplam, developed for treating spinal muscular atrophy. The results quantitatively characterize the specificities of risdiplam and branaplam for 5' splice site sequences, suggest that branaplam recognizes 5' splice sites via two distinct interaction modes, and contradict the prevailing two-site hypothesis for risdiplam activity at SMN2 exon 7. The results also show that anomalous single-drug cooperativity, as well as multi-drug synergy, are widespread among small-molecule drugs and antisense-oligonucleotide drugs that promote exon inclusion. Our quantitative models thus clarify the mechanisms of existing treatments and provide a basis for the rational development of new therapies.