Search

RNA-RNA interactions (RRIs) can dictate RNA molecules to form intricate higher-order structures and bind their RNA substrates in diverse biological processes. To elucidate the function, binding specificity, and regulatory mechanisms of various RNA molecules, especially the vast repertoire of non-coding RNAs, advanced technologies and methods that globally map RRIs are extremely valuable. In the past decades, many state-of-the-art technologies have been developed for this purpose. This review focuses on those high-throughput technologies for the global mapping of RRIs. We summarize the key concepts and the pros and cons of different technologies. In addition, we highlight the novel biological insights uncovered by these RRI mapping methods and discuss the future challenges for appreciating the crucial roles of RRIs in gene regulation across bacteria, viruses, archaea, and mammals. Ye et al. summarize technological advancements in the global mapping of RNA-RNA interactions (RRIs). The authors delve into the core concepts, advantages, and limitations of various technologies, pointing out future challenges and elucidating how these technical innovations have unveiled fresh perspectives on the regulatory functions of RRIs. [ABSTRACT FROM AUTHOR]

The Javan gibbon, Hylobates moloch, is an endangered gibbon species restricted to the forest remnants of western and central Java, Indonesia, and one of the rarest of the Hylobatidae family. Hylobatids consist of 4 genera (Holoock, Hylobates, Symphalangus, and Nomascus) that are characterized by different numbers of chromosomes, ranging from 38 to 52. The underlying cause of this karyotype plasticity is not entirely understood, at least in part, due to the limited availability of genomic data. Here we present the first scaffold-level assembly for H. moloch using a combination of whole-genome Illumina short reads, 10X Chromium linked reads, PacBio, and Oxford Nanopore long reads and proximity-ligation data. This Hylobates genome represents a valuable new resource for comparative genomics studies in primates.

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. 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. 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. [ABSTRACT FROM AUTHOR]

RNA structure has been increasingly recognized as a critical player in the biogenesis and turnover of many transcripts classes. In eukaryotes, the prediction of RNA structure by thermodynamic modeling meets fundamental limitations due to the large sizes and complex, discontinuous organization of eukaryotic genes. Signatures of functional RNA structures can be found by detecting compensatory substitutions in homologous sequences, but a comparative approach is applicable only within conserved sequence blocks. Here, we developed a computational pipeline called PHRIC, which is not limited to conserved regions and relies on RNA contacts derived from RNA in situ conformation sequencing (RIC-seq) experiments. It extracts pairs of short RNA fragments surrounded by nested clusters of RNA contacts and predicts long, nearly perfect complementary base pairings formed between these fragments. In application to a panel of RIC-seq experiments in seven human cell lines, PHRIC predicted ~12,000 stable long-range RNA structures with equilibrium free energy below -15 kcal/mol, the vast majority of which fall outside of regions annotated as conserved among vertebrates. These structures, nevertheless, show some level of sequence conservation and remarkable compensatory substitution patterns in other clades. Furthermore, we found that introns have a higher propensity to form stable long-range RNA structures between each other, and moreover that RNA structures tend to concentrate within the same intron rather than connect adjacent introns. These results for the first time extend the application of proximity ligation assays to RNA structure prediction beyond conserved regions. [ABSTRACT FROM AUTHOR]

RNA structure has been increasingly recognized as a critical player in the biogenesis and turnover of many transcripts classes. In eukaryotes, the prediction of RNA structure by thermodynamic modeling meets fundamental limitations due to the large sizes and complex, discontinuous organization of eukaryotic genes. Signatures of functional RNA structures can be found by detecting compensatory substitutions in homologous sequences, but a comparative approach is applicable only within conserved sequence blocks. Here, we developed a computational pipeline called PHRIC, which is not limited to conserved regions and relies on RNA contacts derived from RNA in situ conformation sequencing (RIC-seq) experiments. It extracts pairs of short RNA fragments surrounded by nested clusters of RNA contacts and predicts long, nearly perfect complementary base pairings formed between these fragments. In application to a panel of RIC-seq experiments in seven human cell lines, PHRIC predicted ~12,000 stable long-range RNA structures with equilibrium free energy below −15 kcal/mol, the vast majority of which fall outside of regions annotated as conserved among vertebrates. These structures, nevertheless, show some level of sequence conservation and remarkable compensatory substitution patterns in other clades. Furthermore, we found that introns have a higher propensity to form stable long-range RNA structures between each other, and moreover that RNA structures tend to concentrate within the same intron rather than connect adjacent introns. These results for the first time extend the application of proximity ligation assays to RNA structure prediction beyond conserved regions.

Improved methods for molecular analyses are obviously central for medical research. I will describe herein our work developing tools to reveal molecular states in health and disease. I will recount how I got started in this endeavor, and how our early work characterizing genetic variation led onto high-throughput protein measurements and to techniques for imaging the distribution of proteins and their activity states in tissues. I will also describe a more recent technique to measure even exceedingly rare genetic variants in order to monitor recurrence of disease for tumor patients.

Tumor cell-derived extracellular vesicles (TEVs) contain numerous cellular molecules and are considered potential biomarkers for non-invasive liquid biopsy. However, due to the low abundance of TEVs secreted by tumor cells and their phenotypic heterogeneity, there is a lack of sensitive and specific methods to quantify TEVs. Here, we developed a dual-aptamer proximity ligation-coupled hybridization chain reaction (HCR) method for tracing TEVs, exploiting CRISPR to achieve highly sensitive detection. Taking advantage of the high binding affinity of aptamers, the two aptamers (Apt EpCAM , Apt HER2) exhibited the high selectivity for TEVs recognition. HCR generated long-repeated sequence containing multiple crRNA targetable barcodes, and the signals were further amplified by CRISPR upon recognizing the HCR sequences, thereby enhancing the sensitivity. Under optimal conditions, the developed method demonstrated a favorable linear relationship in the range of 2 × 103−107 particles/μL, with a limit of detection (LOD) of 3.3 × 102 particles/μL. We directly applied our assay to clinical plasma analysis, achieving 100 % accuracy in cancer diagnosis, thus demonstrating the potential clinical applications of TEVs. Due to its simplicity and rapidity, excellent sensitivity and specificity, this method has broad applications in clinical medicine. [Display omitted] • Dual-aptamers provide high specificity and accurate analysis of TEV. • CRISPR increases the sensitivity and reduces the LOD compared to HCR. • TEV are able to obtain a high accuracy of cancer diagnosis (100 %). [ABSTRACT FROM AUTHOR]

Exosomes, carrying cancer-related proteins derived from parental cells, provide a liquid biopsy probability for early cancer diagnosis. However, the direct analysis of tumor derived exosomes in complex human fluids is still challenging due to the matrix interferences and the low proportion of cancer derived exosomes. Herein, a cholesterol-epithelial cell adhesion molecule (EpCAM) aptamer-mediated proximity ligation assay triggering multicomponent nucleic acid enzyme (MNAzyme) assembly was proposed for detection of exosomes by lanthanide elemental labeling inductively coupled plasma mass spectrometry (ICP-MS). Specifically, the exosomes were recognized through EpCAM aptamer and cholesterol, allowing aptamer EpCAM -partzyme A and cholesterol-partzyme B to hybridize with each other, and assembling into the exosome-MNAzyme complex. With the presence of the cofactor Mn2+, the exosome-MNAzyme complex could catalyze the cleavage of the ribonucleobase-containing substrate deoxyribonucleic acid (DNA) on the Tb-substrate DNA-magnetic beads, releasing Tb tags as readout signal for ICP-MS detection. Due to the recognition of dual exosome surface biomarkers through the proximity probes, the selectivity for the detection of tumor derived exosomes was greatly improved. The developed approach was used for the direct analysis of exosomes in human plasma samples from oral squamous cell carcinoma (OSCC) patients and healthy volunteers, and the concentration of exosomes in the OSCC patient group was found to be significantly higher than that in the healthy group. It manifested that the proposed strategy can easily distinguish healthy volunteers from cancer patients by purification-free quantification of exosomes in human plasma, exhibiting good potential of the developed strategy in early cancer diagnosis. • Cholesterol-EpCAM-mediated proximity ligation assay triggers MNAzyme assembly. • Selectivity for tumor derived exosomes is improved due to the proximity probes. • Elemental labeling ICP-MS is used for direct analysis of exosome in human plasma. [ABSTRACT FROM AUTHOR]

RNA molecules can attach to chromatin. It remains difficult to know what RNAs are associated with chromatin and where the genomic target loci of these RNAs are. Here, we present MARGI (mapping RNA-genome interactions), a technology to massively reveal native RNA-chromatin interactions from unperturbed cells. The gist of this technology is to ligate chromatin-associated RNAs (caRNAs) with their target genomic sequences by proximity ligation, forming RNA-DNA chimeric sequences, which are converted to a sequencing library for paired-end sequencing. Using MARGI, we produced RNA-genome interaction maps for human embryonic stem cells (ESCs) and human embryonic kidney (HEK) cells. MARGI revealed hundreds of caRNAs, including previously known XIST, SNHG1, NEAT1, and MALAT1, as well as each caRNA's genomic interaction loci. Using a cross-species experiment, we estimated that approximately 2.2% of MARGI-identified interactions were false positives. In ESCs and HEK cells, the RNA ends of more than 5% of MARGI read pairs were mapped to distal or inter-chromosomal locations as compared to the locations of their corresponding DNA ends. The majority of transcription start sites are associated with distal or inter-chromosomal caRNAs. Chromatin-immunoprecipitation-sequencing (ChIP-seq)-reported H3K27ac and H3K4me3 levels are positively correlated, while H3K9me3 is negatively correlated, with MARGI-reported RNA attachment levels. The MARGI technology should facilitate revealing novel RNA functions and their genomic target regions.

The nuclear paraspeckle assembly transcript 1 (NEAT1) locus encodes two long non-coding (lnc)RNA isoforms that are upregulated in many tumours and dynamically expressed in response to stress. NEAT1 transcripts form ribonucleoprotein complexes with numerous RNA-binding proteins (RBPs) to assemble paraspeckles and modulate the localisation and activity of gene regulatory enzymes as well as a subset of messenger (m)RNA transcripts. The investigation of the dynamic composition of NEAT1-associated proteins and mRNAs is critical to understand the function of NEAT1. Interestingly, a growing number of biochemical and genetic tools to assess NEAT1 interactomes has been reported. Here, we discuss the Hybridisation Proximity (HyPro) labeling technique in the context of NEAT1. HyPro labeling is a recently developed method to detect spatially ordered interactions of RNA-containing nuclear compartments in cultured human cells. After introducing NEAT1 and paraspeckles, we describe the advantages of the HyPro technology in the context of other methods to study RNA interactomes, and review the key findings in mapping NEAT1-associated RNA transcripts and protein binding partners. We further discuss the limitations and potential improvements of HyPro labeling, and conclude by delineating its applicability in paraspeckles-related cancer research. [ABSTRACT FROM AUTHOR]

Improved methods for molecular analyses are obviously central for medical research. I will describe herein our work developing tools to reveal molecular states in health and disease. I will recount how I got started in this endeavor, and how our early work characterizing genetic variation led onto high-throughput protein measurements and to techniques for imaging the distribution of proteins and their activity states in tissues. I will also describe a more recent technique to measure even exceedingly rare genetic variants in order to monitor recurrence of disease for tumor patients. [ABSTRACT FROM AUTHOR]

N6‐methyladenosine (m6A) modification—the most prevalent mammalian RNA internal modification—plays key regulatory roles in mRNA metabolism. Current approaches for m6A modified RNA analysis limit at bulk‐population level, resulting in a loss of spatiotemporal and cell‐to‐cell variability information. Here we proposed a m6A‐specific in situ hybridization mediated proximity ligation assay (m6AISH‐PLA) for cellular imaging of m6A RNA, allowing to identify m6A modification at specific location in RNAs and image m6A RNA with single‐cell and single‐molecule resolution. Using m6AISH‐PLA, we investigated the m6A level and subcellular location of HSP70 RNA103‐m6A in response to heat shock stress, and found an increased m6A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6AISH‐PLA can serve in the study of m6A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis. [ABSTRACT FROM AUTHOR]

Bacteriophages play important roles in regulating the intestinal human microbiota composition, dynamics, and homeostasis, and characterizing their bacterial hosts is needed to understand their impact. We applied a metagenomic Hi-C approach on 10 healthy human gut samples to unveil a large infection network encompassing more than 6000 interactions bridging a metagenomic assembled genomes (MAGs) and a phage sequence, allowing to study in situ phage-host ratio. Whereas three-quarters of these sequences likely correspond to dormant prophages, 5% exhibit a much higher coverage than their associated MAG, representing potentially actively replicating phages. We detected 17 sequences of members of the crAss-like phage family, whose hosts diversity remained until recently relatively elusive. For each of them, a unique bacterial host was identified, all belonging to different genus of Bacteroidetes. Therefore, metaHiC deciphers infection network of microbial population with a high specificity paving the way to dynamic analysis of mobile genetic elements in complex ecosystems.

Detection of breast cancer-derived exosomes can help provide valuable information for the diagnosis of breast cancer. However, accurate detection of breast cancer exosomes remains challenging. Herein, taking SK-BR-3 exosome, a HER2-positive breast cancer exosome, as target model, a dual-mode assay based on complementary scattering signals for accurate detection of breast cancer-originated exosomes was constructed by proximity ligation mediated RCA reaction. To assure accuracy, three probes recognition (consisting of EpCAM, HER2, and MUC1 aptamers for the recognition of proteins on the surface of exosomes), proximity ligation reaction, and complementary scattering signals produced by the gold nanoparticles (Au NPs) in the supernatant and the magnetic beads-Au NPs (MBs-Au NPs) in the precipitate were used. This assay was equipped to distinguish HER2-positive breast cancer exosomes from HER2-negative and normal exosomes. Due to the RCA reaction triggered by proximity ligation, the dual-mode assay can detect SK-BR-3 exosome with a concentration of 1.0 × 102 particles/μL. With excellent accuracy and sensitivity, the assay can also distinguish healthy individuals from breast cancer patients, making it suitable for clinical application. This work was expected to provide an effective tool for accurate detection of breast cancer-derived exosomes. [Display omitted] • Three probes recognition were used to assure the accuracy. • Proximity ligation was used to improve the accuracy. • The complementary scattering signals further improve the accuracy. • This assay could accurately detect breast cancer exosomes in clinical samples. [ABSTRACT FROM AUTHOR]

Our understanding of RNA structure has lagged behind that of proteins and most other biological polymers, largely because of its ability to adopt multiple, and often very different, functional conformations within a single molecule. Flexibility and multifunctionality appear to be its hallmarks. Conventional biochemical and biophysical techniques all have limitations in solving RNA structure and to address this in recent years we have seen the emergence of a wide diversity of techniques applied to RNA structural analysis and an accompanying appreciation of its ubiquity and versatility. Viral RNA is a particularly productive area to study in that this economy of function within a single molecule admirably suits the minimalist lifestyle of viruses. Here, we review the major techniques that are being used to elucidate RNA conformational flexibility and exemplify how the structure and function are, as in all biology, tightly linked.

Neuroinflammatory diseases, such as Alzheimer’s disease (AD) and traumatic brain injury (TBI), are associated with the extravascular deposition of the fibrinogen (Fg) derivative fibrin and are accompanied with memory impairment. We found that during the hyperfibrinogenemia that typically occurs during AD and TBI, extravasated Fg was associated with amyloid beta and astrocytic cellular prion protein (PrPC). These effects coincided with short-term memory (STM) reduction and neurodegeneration. However, the mechanisms of a direct Fg–neuron interaction and its functional role in neurodegeneration are still unclear. Cultured mouse brain neurons were treated with Fg in the presence or absence of function-blockers of its receptors, PrPC or intercellular adhesion molecule-1 (ICAM-1). Associations of Fg with neuronal PrPC and ICAM-1 were characterized. The expression of proinflammatory marker interleukin 6 (IL-6) and the generation of reactive oxygen species (ROS), mitochondrial superoxide, and nitrite in neurons were assessed. Fg-induced neuronal death was also evaluated. A strong association of Fg with neuronal PrPC and ICAM-1, accompanied with overexpression of IL-6 and enhanced generation of ROS, mitochondrial superoxide, and nitrite as well as the resulting neuronal death, was found. These effects were reduced by blocking the function of neuronal PrPC and ICAM-1, suggesting that the direct interaction of Fg with its neuronal receptors can induce overexpression of IL-6 and increase the generation of ROS, nitrite, and mitochondrial superoxide, ultimately leading to neuronal death. These effects can be a mechanism of neurodegeneration and the resultant memory reduction seen during TBI and AD.

Abstract Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo–NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.

Chemoproteomic methods can report directly on endogenous, active enzyme populations, which can differ greatly from measures of transcripts or protein abundance alone. Detection and quantification of family-wide probe engagement generally requires LC-MS/MS or gel-based detection methods, which suffer from low resolution, significant input proteome requirements, laborious sample preparation, and expensive equipment. Therefore, methods that can capitalize on the broad target profiling capacity of family-wide chemical probes but that enable specific, rapid, and ultrasensitive quantitation of protein activity in native samples would be useful for basic, translational, and clinical proteomic applications. Here we develop and apply a method that we call soluble activity-dependent proximity ligation (sADPL), which harnesses family-wide chemical probes to convert active enzyme levels into amplifiable barcoded oligonucleotide signals. We demonstrate that sADPL coupled to quantitative PCR signal detection enablesmultiplexed “writing” and “reading” of active enzyme levels across multiple protein families directly at picogram levels of whole, unfractionated proteome. sADPL profiling in a competitive format allows for highly sensitive detection of drug–protein interaction profiling, which allows for direct quantitative measurements of in vitro and in vivo on- and offtarget drug engagement. Finally, we demonstrate that comparative sADPL profiling can be applied for high-throughput molecular phenotyping of primary human tumor samples, leading to the discovery of new connections between metabolic and proteolytic enzyme activity in specific tumor compartments and patient outcomes. We expect that this modular and multiplexed chemoproteomic platform will be a general approach for drug target engagement, as well as comparative enzyme activity profiling for basic and clinical applications. [ABSTRACT FROM AUTHOR]

Genome structural variations, including duplications, deletions, insertions, and inversions, are central in the evolution of eukaryotic genomes. However, structural variations present challenges for high-quality genome assembly, hampering efforts to understand the evolution of gene families and genome architecture. An example is the genome of the pea aphid (Acyrthosiphon pisum) for which the current assembly is composed of thousands of short scaffolds, many of which are known to be misassembled. Here, we present an improved version of the A. pisum genome based on the use of two long-range proximity ligation methods. The new assembly contains four long scaffolds (40–170 Mb), corresponding to the three autosomes and the X chromosome of A. pisum , and encompassing 86% of the new assembly. Assembly accuracy is supported by several quality assessments. Using this assembly, we identify the chromosomal locations and relative ages of duplication events, and the locations of horizontally acquired genes. The improved assembly illuminates the mode of gene family evolution by providing proximity information between paralogs. By estimating nucleotide polymorphism and coverage depth from resequencing data, we determined that many short scaffolds not assembling to chromosomes represent hemizygous regions, which are especially frequent on the highly repetitive X chromosome. Aligning the X-linked aphicarus region, responsible for male wing dimorphism, to the new assembly revealed a 50-kb deletion that cosegregates with the winged male phenotype in some clones. These results show that long-range scaffolding methods can substantially improve assemblies of repetitive genomes and facilitate study of gene family evolution and structural variation. [ABSTRACT FROM AUTHOR]

Transcriptional regulation involves a series of sophisticated protein–protein and protein–DNA interactions (PPI and PDI). Some transcriptional complexes, such as c‐Fos/c‐Jun and their binding DNA fragments, have been conserved over the past one billion years. Considering the thermodynamic principle for transcriptional complex formation, we hypothesized that the c‐Fos/c‐Jun complex may represent a thermodynamic summit in the evolutionary space. To test this, we invented a new method, termed One‐Pot‐seq, which combines cDNA display and proximity ligation to analyse PPI/PDI complexes simultaneously. We found that the wild‐type c‐Fos/c‐Jun complex is indeed the most thermodynamically stable relative to various mutants of c‐Fos/c‐Jun and binding DNA fragments. Our method also provides a universal approach to detect transcriptional complexes and explore transcriptional regulation mechanisms. [ABSTRACT FROM AUTHOR]

Highlights • A high-throughput imaging method for visualization of protein-protein interactions. • A panel of 60 lamin A/C interactors were identified. • Progerin alters interaction behavior of a subset of nuclear proteins. • Changes in protein interactions largely correlate with interactor expression. Abstract Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (hi gh-throughput imaging p roximity l igation a ssay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Screened for substitutions in the biotin binding site that modulate BirA activity. • An Arg-to-Lys substitution at position 118 increases biotinoyl-5′-AMP release. • Used methods to discriminate between self- and trans -biotinylation by BirA. • PCNA was identified as a Lamin A-proximal protein using BirA (Arg118Lys). Abstract The discovery and validation of protein–protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest. Here, we explore how amino acid substitutions at Arg118 affect the biochemical properties of BirA. We found that relative to wild-type BirA, the Arg118Lys substitution both slightly reduced biotin affinity and increased the release of reactive biotinyl-5′-AMP. BioID using a BirA(Arg118Lys)-Lamin A fusion enabled identification of PCNA as a lamina-proximal protein in HEK293T cells, a finding that was validated by immunofluorescence microscopy. Our data expand on the concept that proximity labeling by BirA fused to proteins of interest can be modulated by amino acid substitutions that affect biotin affinity and the release of biotinyl-5′-AMP. [ABSTRACT FROM AUTHOR]

Proteins often exist and function as part of higher-order complexes or networks. A challenge is to identify the universe of proximal and interacting partners for a given protein. We describe how the high-activity promiscuous biotin ligase called TurboID is fused to the actin-binding peptide LifeAct to label by biotinylation proteins that bind, or are in close proximity, to actin. The rapid enzyme kinetics of TurboID allows the profiles of actin-binding proteins to be compared under different conditions, such as acute disruption of filamentous actin structures with cytochalasin D., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Highlights • PLiRT-PCR is a sensitive immunoassay that combines antibody-oligo conjugates, enzymatic ligation, and PCR for quantitative protein detection. • We show that PLiRT-PCR assay enables detection of semen as well as sperm cells from simulated sexual assault samples. • The assay processes multiple samples at a time and utilizes existing instruments in forensic labs. • Employing PLiRT-PCR assay in routine forensic work may reduce backlogged samples and help meet the challenges of serological testing. Abstract The positive identification of seminal fluids in sexual assault crimes is considered crucial evidence to determine whether a sexual act occurred or not. However, current presumptive methods lack specificity and sensitivity. Confirmation of semen by microscopic examination of spermatozoa is laborious, time consuming, and can sometimes lead to negative or inconclusive results. Here we report the use of the Proximity Ligation Real-Time PCR (PLiRT-PCR) assay as an attractive and promising confirmatory method for the identification of semen and sperm proteins using two polyclonal antibodies, Prostate Specific Antigen (PSA) and Sperm-Specific Protein (SP10), respectively. PLiRT-PCR, relies on protein recognition by pairs of proximity probes (antibody-DNA conjugates) that give rise to a ligated DNA strand. The ligated DNA strand is then amplified and detected by qPCR. [ABSTRACT FROM AUTHOR]

A novel method for study the interaction between carbohydrate and lectin was developed in homogenous solution. DNA was introduced to the recognition process of carbohydrate with protein. Due to the versatile detection mode of DNA, the detection efficiency for carbohydrate and protein was highly promoted. Herein, two kinds of mannose-DNA conjugates were synthesized. One of which was used to prepare DNA-templated silver nanocluster (AgNCs) and the other one contained guanine-rich DNA sequences. When the mannose of two conjugates binding to lectin Concanavalin A (Con A), great enhancement on fluorescence intensity was obtained due to the proximity ligation of the DNA-templated Ag NCs with guanine-rich DNA sequences. Hence Con A can be quantified conveniently in homogenous solution using Ag NCs with low toxicity and tunable fluorescence. Moreover, carbohydrate functionalized DNA-templated AgNCs was also utilized for cancer cell imaging based on the recognition of mannose with mannose receptor on cancer cell MDA-MB-231. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play a significant role in regulating diverse physiological processes, and are regarded as novel diagnostic biomarkers. However, the sensitive and reliable miRNA detection remains a huge challenge. Herein, we propose a proximity ligated initiated magnesium ion (Mg2+)-dependent DNAzyme-powered signal cascade for sensitive, accurate and reliable detection of miRNAs. Three signal amplification processes are involved in this approach, including the target miRNA recycle, DNAzyme powered substrate cleavage, and catalytic hairpin reaction (CHA). Based on this, the approach shows a low limit of detection of 523 aM and a wide detection range of 7 orders of magnitudes, which is comparable or superior to most of the former miRNA detection methods. In addition, the approach also possesses a high selectivity to target miRNA, suggesting a potential promising future of the approach for rapid detection of miRNAs in the application of developing novel tools for skin cancer diagnosis, and recovery evaluation. [Display omitted] • Sensitive and reliable detection of miRNAs is highly-desired. • We propose a proximity ligated initiated magnesium ion (Mg2+)-dependent DNAzyme-powered signal cascade strategy for miRNA detection. • Target mediated proximity ligation endows the method a high specificity. • The approach shows a low limit of detection of 523 aM and a wide detection range of 7 orders of magnitudes. [ABSTRACT FROM AUTHOR]

The synthetic biology has employed the synthetic gene networks through engineering to construct various functions in biological systems. However, the use of gene circuits to create sensors for detecting low-abundance targets has been limited due to the lack of signal amplification strategies beyond direct output of detection signals. To address this issue, we introduce a novel method utilizing S elective R ecognition P roximity L igation and signal amplification with T7 Tra nscription and C RISPR/Cas12a s ystem (SRPL-TraCs), which permits the incorporation of cell-free gene circuits with signal amplification and enables the construction of high-order cascade signal amplification strategy to detect biomarkers in homogeneous systems. Specifically, the SRPL-TraCs utilizes selective recognition proximity ligation with high-fidelity T4 DNA ligase and generates a unique crRNA via T7 transcription, along with target-activated Cas12a/crRNA system to achieve excellent specificity for HIV-1 DNA. With this straightforward synthetic biology-based method, the proposed SRPL-TraCs has the potential to detect numerous other interesting targets beyond the nucleic acids. [Display omitted] • A SRPL-TraCs was developed for the biomarker detection in homogeneous system. • The Selective Recognition Proximity Ligation guaranteed the specificity of SRPL-TraCs. • The T7 Transcription and CRISPR/Cas12a system was applied for improving the detection performance. • The incorporation of synthetic biology method with signal amplification could extend to other biomarker detection. [ABSTRACT FROM AUTHOR]

Here, we have developed a facile fluorometric system for the detection of adenosine triphosphate (ATP) by a rolling circle amplification (RCA) based on proximity ligation mediated amplification, and simultaneously achieved the release of the anticancer drug doxorubicin (DOX) through the mesoporous silicon system. Once the ATP molecule is present, the linker DNA will be released from the graphene oxide (GO) surface and hybridized to the template DNA of the GO surface joining with ligation enzyme. RCA reaction is followed by the addition of the phi29 DNA polymerase. The product of RCA reaction contains a base fragment complementary to the signal DNA, allowing the fluorescent oligonucleotide probe to be released from the GO surface and fluorescence is recovered. The strong fluorescence signal realized the sensitive detection of ATP. Gate DNA were modified to the surface of the mesoporous silica (MSN) by electrostatic attraction to encapsulate DOX. After the above-mentioned RCA process, its result that long DNA chain containing a base fragment complementary to gate DNA, would be hybridized to the gate DNA strand on the surface of MSN, which opened the MSN hole and released the drug DOX into cell for HeLa cell therapy. And the specificity to folate receptor overexpressed on cell surface was satisfactory which would be beneficial for cancer therapy. [ABSTRACT FROM AUTHOR]

Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo-NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976. [ABSTRACT FROM AUTHOR]

Background: We previously reported that the expression of fibroblast growth factor receptor-like 1 (FGFRL1) was associated with the progression of esophageal squamous cell carcinoma (ESCC). However, the clinical roles of FGFR1, FGFR2, FGFR3, and FGFR4 in ESCC have not yet been examined in detail. Furthermore, it currently remains unclear why FGFRL1 is a prognostic factor for ESCC. Materials and methods: A squamous cell carcinoma tissue microarray established in Toyama University was used to evaluate the expression of the 4 FGFRs. Sixty-nine ESCC specimens were obtained from patients who had undergone surgery between 1990 and 2008. The immunohistochemical results obtained herein were compared to those for FGFRL1 from our previous study. We also determined whether FGFRL1 bound to other FGFRs that were selected using the tissue microarray analysis. Results: The frequency of different FGFR overexpression combinations revealed that FGFRL1, FGFR1, and FGFR4 were the predominant types. In contrast to FGFRL1, no association was observed between the expression of each FGFR and patient prognosis. Regarding the combination analysis, patients that co-expressed FGFRL1 and FGFR1 had the worst prognosis, while patients who tested negative for the expression of both FGFRL1 and FGFR4 had the best prognosis. The results of the proximity ligation assay demonstrated that FGFRL1 bound to FGFR1 and FGFR4. Conclusions: Of the 5 FGFRs examined, the expression of FGFRL1 was the main prognostic factor in ESCC patients. FGFR1 and FGFR4 may be sub-driving factors of ESCC as well as the main targets of the heterodimer of FGFRL1. [ABSTRACT FROM AUTHOR]

Background: There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. Results: We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment. Conclusions: We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain-ligand interactions which could have wide-ranging applications in both basic and translational cancer research. [ABSTRACT FROM AUTHOR]

Peroxisomes are essential organelles in mammals, which contribute to cellular lipid metabolism and redox homeostasis. They do not function as isolated entities but cooperate and interact with other subcellular organelles, in particular the endoplasmic reticulum, mitochondria, and lipid droplets. Those interactions are often mediated by membrane contact sites. Tether proteins at those sites bring the organelles in close proximity to facilitate metabolite and lipid transfer as well as organelle communication. There is great interest in the investigation of the physiological functions of peroxisome-organelle contacts and how they are regulated. Here, we present an antibody- and fluorescence-based proximity ligation approach used successfully in our laboratory for the detection and quantification of peroxisome-organelle interactions in cultured mammalian cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chromosome conformation capture technology and its derivatives have been widely used to study genome organization. Among them, Hi-C (chromosome conformation capture coupling with high-throughput sequencing) is popular in dissecting chromatin architecture on the genome-wide level. However, the intrinsic limitations prevent its application when it comes to rare samples. Here, we present easy Hi-C, a biotin-free technology that dramatically reduces DNA loss and is suitable for low-input samples., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

High-throughput RNA proximity ligation assays are molecular methods that are used to simultaneously analyze the spatial proximity of many RNAs in living cells. Their principle is based on cross-linking, fragmentation, and subsequent religation of RNAs, followed by high-throughput sequencing. The generated fragments have two different types of splits, one resulting from pre-mRNA splicing and the other formed by the ligation of spatially close RNA strands. Here, we present RNAcontacts, a universal pipeline for detecting RNA-RNA contacts in high-throughput RNA proximity ligation assays. RNAcontacts circumvents the inherent problem of mapping sequences with two distinct types of splits using a two-pass alignment, in which splice junctions are inferred from a control RNA-seq experiment on the first pass and then provided to the aligner as bona fide introns on the second pass. Compared to previously developed methods, our approach allows for a more sensitive detection of RNA contacts and has a higher specificity with respect to splice junctions that are present in the biological sample. RNAcontacts automatically extracts contacts, clusters their ligation points, computes the read support, and generates tracks for visualizing through the UCSC Genome Browser. The pipeline is implemented in Snakemake, a reproducible and scalable workflow management system for rapid and uniform processing of multiple datasets. RNAcontacts is a generic pipeline for the detection of RNA contacts that can be used with any proximity ligation method as long as one of the interacting partners is RNA. RNAcontacts is available via the GitHub repository https://github.com/smargasyuk/ RNAcontacts/., (Copyright ® 2023 National Research University Higher School of Economics.)

Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids., (© 2023. The Author(s).)

Since Jacob and Monod's characterization of the role of DNA elements in gene control, it has been recognized that the linear organization of genome structure is important for the regulation of gene transcription and hence the manifestation of phenotypes. Similarly, it has long been hypothesized that the spatial organization (in three dimensions evolving through time), as part of the epigenome, makes a significant contribution to the genotype-phenotype transition. Proximity ligation assays commonly known as chromosome conformation capture (3C) and 3C based methodologies (e.g., GCC, HiC, and ChIA-Pet) are increasingly being incorporated into empirical studies to investigate the role that three-dimensional genome structure plays in the regulation of phenotype. The apparent simplicity of these methodologies--crosslink chromatin, digest, dilute, ligate, detect interactions--belies the complexity of the data and the considerations that should be taken into account to ensure the generation and accurate interpretation of reliable data. Here we discuss the probabilistic nature of these methodologies and how this contributes to their endogenous limitations. [ABSTRACT FROM AUTHOR]

The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy. [ABSTRACT FROM AUTHOR]

Non-central nervous system hemangiopericytoma (HPC) and solitary fibrous tumor (SFT) are considered by pathologists as two variants of a single tumor entity now subsumed under the entity SFT. Recent detection of frequent NAB2-STAT6 fusions in both, HPC and SFT, provided additional support for this view. On the other hand, current neuropathological practice still distinguishes between HPC and SFT. The present study set out to identify genes involved in the formation of meningeal HPC. We performed exome sequencing and detected the NAB2-STAT6 fusion in DNA of 8/10 meningeal HPC thereby providing evidence of close relationship of these tumors with peripheral SFT. Due to the considerable effort required for exome sequencing, we sought to explore surrogate markers for the NAB2-STAT6 fusion protein. We adopted the Duolink proximity ligation assay and demonstrated the presence of NAB2-STAT6 fusion protein in 17/17 HPC and the absence in 15/15 meningiomas. More practical, presence of the NAB2-STAT6 fusion protein resulted in a strong nuclear signal in STAT6 immunohistochemistry. The nuclear reallocation of STAT6 was detected in 35/37 meningeal HPC and 25/25 meningeal SFT but not in 87 meningiomas representing the most important differential diagnosis. Tissues not harboring the NAB2-STAT6 fusion protein presented with nuclear expression of NAB2 and cytoplasmic expression of STAT6 proteins. In conclusion, we provide strong evidence for meningeal HPC and SFT to constitute variants of a single entity which is defined by NAB2-STAT6 fusion. In addition, we demonstrate that this fusion can be rapidly detected by STAT6 immunohistochemistry which shows a consistent nuclear reallocation. This immunohistochemical assay may prove valuable for the differentiation of HPC and SFT from other mesenchymal neoplasms. [ABSTRACT FROM AUTHOR]

Aims: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. Methods: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. Results: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. Conclusions: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours. [ABSTRACT FROM AUTHOR]