Biology subjects

Tan, W.

Publications and source records attributed to Tan, W..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Single-vessel transcriptome map pathological landscapes and reveal NR2F2-mediated smooth muscle cell phenotype acquisition in capillary malformations

BackgroundCapillary malformation (CM) is a congenital vascular anomaly affecting the skin, mucosa, and brain, yet the understanding of its vascular pathogenesis remains limited. MethodsWe applied spatial whole-transcriptome profiling (GeoMx) and gene set enrichment analysis within CM lesions at single vasculature level. Differentially expressed genes were validated by immunofluorescence staining. Phosphoproteomics was profiled to uncover lesion- wide phosphorylation sites on proteins. Single-cell RNA sequencing was performed on CM- derived induced pluripotent stem cells (iPSCs) to determine differentiation trajectories of lesional vascular lineages. In silico gene perturbation was used to predict candidate genes for modulating vascular pathological progression, followed by functional validation in CM iPSC-derived endothelial cells (ECs) using a Tet-on system. ResultsA spatial transcriptomic atlas was constructed, and pathological landscape of individual CM vasculature was delineated. CM vessels exhibited hallmarks of endothelial-to-mesenchymal transition (EndMT), including disruption of adherens junctions (AJs), vascular identity transitions, and metabolic remodeling. Phosphoproteomics confirmed that differentially phosphorylated proteins were enriched in EndMT- and AJ-related pathways. Aberrant expression of venous transcriptional factor NR2F2 was observed in lesional ECs and correlated with progressive enlargement from capillaries to larger-caliber vessels containing multiple layers of smooth muscle cells (SMCs). In CM iPSCs, differentiation course yielded reduced ECs but increased SMCs. In silico knockout simulation predicted NR2F2 as a crucial regulator of facilitating SMC phenotype in CM. Consistently, enforced NR2F2 expression during iPSC differentiation suppressed endothelial markers while inducing SMC-associated genes. ConclusionsSingle CM vasculature displays pathological hallmarks characterized by EndMT and AJ disruption, leading to progressive vascular remodeling. NR2F2 functions as a central regulatory factor orchestrating the acquisition of the SMC phenotype, thereby representing a potential therapeutic target in CM.

pathology↗

bioRxiv2025

RNA structural heterogeneity in a eukaryotic cell influences its heat shock response

RNA structure-based gene regulation remains under-explored in eukaryotes. While RNA can form different conformations in solution, the extent to which it folds into structure ensembles and how the different conformations regulate gene expression still needs to be fully understood. We coupled the SHAPE compound NAI-N3 with direct RNA sequencing to identify structure modifications along a single RNA molecule (sm-PORE-cupine). Using a combination of base mapping and direct signal alignment, we boosted the percentage of mappable RNA molecules from direct RNA sequencing. Using Bernoulli Mixture Model (BMM) clustering, we show that we can separate RNA structure ensembles from ligand-bound and unbound riboswitches accurately, identify isoform-specific structure ensembles along the SARS-CoV-2 genome, and determine RNA structure ensembles in the transcriptome of a eukaryote, C. albicans, at yeast (30{degrees}C) and hyphae (37{degrees}C) states. We observed that RNAs are more structurally homogenous at 37{degrees}C compared to 30{degrees}C, are more variable in vivo than in vitro, and show higher homogeneity in 3UTRs than in the coding region. We also identified structure ensembles that are associated with changes in translation efficiency and decay in C. albicans at 30{degrees}C and 37{degrees}C and validated translational changes using reporter assays. Our work shows that single-molecule RNA structure probing using direct RNA sequencing can be applied to diverse transcriptomes to study the complexity and function of RNA structures.

molecular biology↗

bioRxiv2025

Follistatin-like protein 1 interacts with programmed cell death 4 to promote vascular mimicry in hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling pathway

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is highly prone to recurrence and metastasis after curative treatment. However, the molecular mechanisms governing these post-management setbacks remain largely unknown. Previous studies found that the high expression of follistatin-like protein 1 (FSTL1) in tumors is implicated in recurrence, metastasis, and poor patient prognosis. In this study, we aimed to investigate the role of FSTL1 in HCC. We found that FSTL1 overexpression was positively correlated with vascular mimicry and poor prognosis. In vitro experiments confirmed that the interaction between FSTL1 and programmed cell death 4 (PDCD4) activates epithelial-to-mesenchymal transition and promotes the proliferation, invasion, and migration of liver cancer cells. The protein-protein interaction between FSTL1 and PDCD4 significantly promotes VM as well as the expression of the pro-angiogenic vascular endothelial growth factor by activating AKT/mTOR signaling. Our data suggest that FSTL1 may be a potential target for the treatment of HCC. Implications: Our findings indicate that the protein FSTL1 may be a novel therapeutic target for managing patients with HCC.

cancer biology↗

bioRxiv2025

Embryonic stem cell factors DPPA2/4 facilitate a unique chromatin state in non-small cell lung cancer

Embryonic regulators are often re-expressed in cancers, however the functional and molecular significance of this is not always understood. The epigenetic priming factors Developmental Pluripotency Associated 2 and 4 (DPPA2/4) have crucial roles in early development and are implicated in cancer pathogenesis. We reveal in non-small cell lung cancer (NSCLC), DPPA2/4 co-expression is associated with poorly differentiated tumours, impaired patient outcomes and accelerated in vivo xenograft tumour growth. Proteomic analyses reveal DPPA2/4 dimerise to enhance their protein stability and binding efficiency to nucleosomes. Our multiomic epigenomic analysis uncovered novel functions for DPPA2/4 in facilitating a unique chromatin landscape in NSCLC cells at active promoters and enhancers. These domains are simultaneously marked by active H3K4me3, PRC1 deposited H2AK119Ub but devoid of PRC2 deposited H3K27me3. This paradoxical uncoupling between the activity of the Polycomb Repressive Complexes is likely driven by H3K27ac at these regions that prevents PRC2 catalytic activity but not recruitment. Our results demonstrate how in NSCLC cells, DPPA2/4 promote a "poised for repression" chromatin state, uncoupling complex recruitment from catalytic activity. Our study highlights how aberrant re-activation of embryonic factors in cancers may take on new functions, promoting tumourigenesis.

molecular biology↗

bioRxiv2025

SpatioCell: A Deep Learning Algorithm for High-resolution Single-cell Mapping through Deep Integration of Histology Image and Sequencing Data

Spatial transcriptomics (ST) is a powerful assay to capture gene expression in tissue context. However, due to the limitation of resolution, most existing ST datasets remain at multicellular resolution which hinders comprehensive understanding of the spatial organization. We propose SpatioCell, a computational algorithm to automatically extract both cell type and expression information at single-cell resolution from ST data, through a morpho-transcriptomic spatial reconstruction framework solved via dynamic programming, integrating morphological and transcriptomic information. This framework enables deterministic single-cell spatial reconstruction, assigning cell identities to precise locations rather than inferring spot-level cell-type composition and expression, and further uncovers overlooked microenvironment features while correcting deconvolution errors. Using the ST data from triple negative breast cancer as an example, SpatioCell reveals the significance of the distance between cancer-associated fibroblast (CAF) and tumor or immune cells for disease progression. The establishment of SpatioCell will broaden the biomedical applications of ST and facilitate investigations of single-cell spatial organization.

bioinformatics↗

bioRxiv2025

Predicting PROTAC-targeted Degradation and Designing Androgen Receptor Degraders with AiPROTAC

Proteolysis-targeting chimeras (PROTACs), a pioneering class of heterobifunctional ligands, have emerged as transformative tools in combating cancer and immune-related diseases due to their ability to target previously undruggable proteins and overcome drug resistance. Accurate assessment of the degradation potential of PROTACs is essential for advancing their therapeutic applications. However, the development of robust predictive models is hindered by the scarcity of large-scale datasets and domain-specific tools, as well as the underutilization of existing unlabeled data. To address these challenges, we developed an innovative method called AiPROTAC to predict the degradation capacity of PROTACs. In particular, AiPROTAC integrates graph augmentation, message-passing enhanced encoders and cross-attention mechanisms within a contrastive learning framework. Moreover, AiPROTAC leverages a curated specialized dataset combined with the PROTAC-DB dataset to enhance prediction accuracy. Experimental results across two datasets and six evaluation metrics demonstrate that AiPROTAC consistently outperforms state-of-the-art models. Further case studies underline its superior sensitivity and reliability. Notably, AiPROTAC facilitated the design of a novel androgen receptor (AR) degrader, PROTAC GT19, which achieved enhanced in vitro degradation compared to Bavdegalutamide (ARV-110). This advancement highlights our AiPROTACs potential to accelerate PROTAC-based therapeutic development and optimization.

bioinformatics↗

bioRxiv2025

Electromotility can be disassociated from gating charge movement in outer hair cells of conditional alpha2 spectrin knockout mice.

Electromotility in mammalian outer hair cells (OHC) is the mechanism underlying cochlear amplification. It is brought about by the piezoelectric-like property of the membrane protein prestin (Slc26a5) that lies in the OHCs lateral plasma membrane. Prestin connects to an underlying cytoskeletal network of circumferential actin filaments that bridge longitudinal spectrin filaments. This network, in turn, lies between the plasma membrane and a closely apposed ER-like tubular array of subsurface cisternae (SSC). Two previous papers examining spectrin knockouts in embryonic hair cells were confined to analyzing the effects on the apical cuticular plate and overlying stereocilia. In this paper, we examine the effects of conditional knockouts of alpha2 spectrin in postnatal OHCs. We find a significant auditory phenotype likely due to the novel disassociation of prestins gating charge movement from OHC electromotility. In addition, OHCs show enlargement in their SSC and plasma membrane-SSC space with preserved cuticular plates and overlying stereocilia, which contrasts with the findings in embryonic knockouts.

physiology↗

bioRxiv2025

Cycling Molecular Assemblies for Selective Cancer Cell Golgi Disruption

The Golgi apparatus is a critical organelle responsible for intracellular trafficking and signaling, orchestrating essential processes such as protein and lipid sorting1-5. Dysregulation of its function has been implicated in various pathologies, including obesity, diabetes, and cancer, highlighting its importance as a potential therapeutic target. Despite this, the development of tools to selectively target the Golgi in specific cell types remain a significant unmet challenge in imaging and drug discovery. Golgi-specific enzyme activities, such as those mediated by protein acyltransferases and thioesterases6, offer an untapped opportunity to develop subcellularly localized therapeutics. Current approaches predominantly rely on direct protein binding but lack the necessary cell selectivity7, underscoring the unmet need for innovative strategies to selectively disrupt Golgi function in cancer cells. Here, we report the development of cycling molecular assemblies (CyMA), a novel class of small peptide derivatives (e.g., dipeptides), which exploit the unique enzymatic environment of the Golgi to establish futile cycles of reversible S-acylation. These assemblies selectively accumulate in cancer cell Golgi, interfering with protein S-acylation cycles and disrupting organelle homeostasis. CyMA impair key Golgi functions, including protein trafficking, glycosylation, and secretion, while demonstrating selective sparing hepatocytes and immune cells such as M1 macrophages. This selective activity represents a paradigm shift, utilizing an enzyme switch and leveraging intracellular environment rather than direct protein binding. Unlike conventional approaches, CyMA reduce tumor growth, drug resistance, and metastasis by pleiotropically disrupting Golgi related functions. By demonstrating the potential of futile cycles as a therapeutic strategy8, this study introduces a generalizable method for targeting organelle-specific enzyme activities. These findings not only underscore the therapeutic potential of CyMA in cancer but also pave the way for future applications in other Golgi-associated diseases.

biochemistry↗

bioRxiv2024

A CRISPR/SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus

As members of the -proteobacteria group, Caulobacter crescentus and its relatives are known for their asymmetric life cycle and comprehensive applications in gene delivery, agricultural biotechnology, and the production of high-value compounds. However, genetic manipulations of these bacteria are often time-consuming and labor-intensive due to the lack of efficient genome editing tools. Here, we report a practical CRISPR/SpCas9M-reporting system that overcomes the limitations of SpCas9 expression, enabling efficient, markerless, and rapid genome editing in C. crescentus. As a demonstration, we successfully knocked out two genes encoding the scaffold proteins, achieving apparent editing efficiencies up to 80%. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized to enhance the editing efficiency or decrease the cell lethality. A nearly zero off-target ratio was observed after the curing of the editor plasmid in editing strains. Furthermore, we applied the CRISPR/SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and reliable editing strategy. We anticipate that this system could be applied to other hard-to-edit organisms, accelerating both basic and applied research in -proteobacteria.

bioengineering↗

bioRxiv2024

Targeting long non-coding RNA RP11-502I4.3 delays the trend of angiogenesis in diabetic retinopathy

Based on our previous findings, we hypothesized that the long non-coding RNA RP11-502I4.3 may be involved in angiogenesis associated with Diabetic retinopathy (DR). We investigated the role of RP11-502I4.3 in DR by examining its regulation of vascular endothelial growth factor (VEGF). We assessed differences in RP11-502I4.3 expression between the normal control group and streptozotocin-induced diabetic rats or high glucose (HG)-stimulated human retinal microvascular endothelial cells (HRMECs). VEGF expression was measured with and without lentiviral vectors overexpressing RP11-502I4.3. We analyzed the structural and functional alterations related to DR. Our analysis revealed that RP11-502I4.3 expression was lower in the retinas of diabetic rats and in HG-stimulated HRMECs compared with normal glucose conditions. Overexpressing of RP11-502I4.3 resulted in decreased VEGF levels. Diabetic rats exhibited retinopathy characterized by thinning of the retinal layer thickness, structural changes in the inner and outer nuclear layers, a reduced count of retinal ganglion cells, and the presence of acellular capillaries. The proliferative activity, migration count, and tube formation ability of HG-treated HRMECs were significantly higher than those of the normal control group; however, these changes were delayed by RP11-502I4.3 overexpression. RP11-502I4.3 downregulation in DR appears to promote angiogenesis.

zoology↗

bioRxiv2024

Intraperitoneal programming of tailored CAR macrophages via mRNA-LNP to boost cancer immunotherapy

Therapeutic strategies for peritoneal metastasis in solid tumors are urgently needed in the clinic. Programming chimeric antigen receptor macrophages (CAR-Ms) in situ offers opportunities for an unmet demand. However, potential intracellular domains (ICDs) for CAR design and their antitumor mechanisms for macrophage empowerment remain to be explored systematically. By developing a targeted mRNA-LNP delivery system for macrophages, we have investigated 36 CAR combinations to determine the impact of CAR-Ms on immune regulation in vitro and in vivo. In two solid tumor mouse models, intraperitoneal programming of CAR-Ms was shown to elicit robust adaptive immune activation and significantly synergize with PD-1/L1 therapy. Single-cell RNA sequencing (scRNA-seq) analysis revealed that CAR-Ms could reshape the immunosuppressive tumor microenvironment (TME) and boost the TCF1+PD-1+ progenitor- exhausted CD8+ T cells (Tpex) population. Meanwhile, we found that tailored CAR-M with CD3{zeta}/TLR4 ICDs could favorably maintain proinflammatory phenotype and simultaneously upregulate MHC I and PD-L1 expression by perturbing NF-{kappa}B pathways. Moreover, the synergism between macrophage PD-L1 knockdown and CAR-M therapy highlighted the need to block the PD-1/L1 axis in antigen cross-presentation. In short, we developed an mRNA-LNP delivery system for intraperitoneal programming of tailored CAR-Ms in vivo and broadened understanding of both regulatory and feedback mechanisms for CAR-M therapies against solid tumors.

bioengineering↗

bioRxiv2024

PepCleav: a classification method for evaluating the cleavability of peptide fragments presented by MHC class I

Neoantigens, due to their tumor specificity and lack of central tolerance, are promising targets for cancer immunotherapy. Evaluating the cleavability of peptide fragments generated by proteasomes and aminopeptidases is crucial for neoantigen identification, but tools for this purpose are scarce. To address this, we developed PepCleav, a method for evaluating peptide cleavability by classifying the amino acids at the proteasome and aminopeptidase recognition sites of the C- and N-termini. By integrating C- and N-terminal cleavability, PepCleav accurately predicted the overall cleavability of peptide fragments in test datasets, correctly identifying 84-89% of cleavable peptides and 63-92% of non-cleavable peptides. We also found that highly cleavable peptides have a higher likelihood of being effective neoantigens, highlighting PepCleavs potential to improve neoantigen identification. PepCleavs source code is publicly available at https://github.com/Dulab2020/PepCleav.

bioinformatics↗

bioRxiv2024

MORC2 phosphorylation fine tunes its DNA compaction activity

Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.

molecular biology↗

bioRxiv2024

Molecular Architecture of Monkeypox Mature Virus

Since the 2022 mpox outbreak, MPXV has caused [~]100,000 infected cases with [~]200 deaths across 117 countries. and was declared a public health emergency of international concern by the WHO. The causative pathogen, monkeypox virus (MPXV), is a zoonotic double-stranded DNA orthopoxvirus. To investigate the structures and replication mechanism of MPXV, the MPXV recombinant proteins and vaccinia virus (VACV) particles have been studied by cryo-EM, however, in situ structural insights into major structural proteins and assembly of the authentic MPXV particles are missing. In this work, we isolated two MPXV strains from the first imported mpox case and the first local mpox case in mainland China. From the two strains, we propagated and purified mature viruses, and applied cryo-electron tomography (cryo-ET) and sub-tomogram averaging (STA) to unveil the architecture of intact MPXV virus. Interestingly, we also discovered irregularly-shaped mature virions with distorted core walls. To our knowledge, these in situ structural insights in MPXV have not been reported before.

biophysics↗

bioRxiv2024

A genome resource for the marine annelid Platynereis dumerilii

The marine annelid Platynereis dumerilii is a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes of P. dumerilii (laboratory culture reference and a single individual assembly) and of the closely related P. massiliensis and P. megalops (single individual assembly) to facilitate comparative genomic approaches and help explore Platynereis biology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of [~]1.47 Gbp for P. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 105 miRNA loci. We further explore the high genetic variation ([~]3% heterozygosity) within the Platynereis species complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development of Platynereis genomic resources.

genomics↗

bioRxiv2024

A Single-Short Partial Reprogramming of the Endothelial Cells decreases Blood Pressure via attenuation of EndMT in Hypertensive Mice

BackgroundSmall artery remodeling and endothelial dysfunction are hallmarks of hypertension. Growing evidence supports a likely causal association between cardiovascular diseases and the presence of endothelial-to-mesenchymal transition (EndMT), a cellular transdifferentiation process in which endothelial cells (ECs) partially lose their identity and acquire additional mesenchymal phenotypes. EC reprogramming represents an innovative strategy in regenerative medicine to prevent deleterious effects induced by cardiovascular diseases. MethodsUsing a partial reprogramming of ECs, via overexpression of Oct-3/4, Sox-2, and Klf-4 (OSK) transcription factors, we aimed to bring ECs back to a youthful phenotype in hypertensive mice. Primary ECs were infected with lentiviral vectors (LV) containing the specific EC marker cadherin 5 (Cdh5) and the fluorescent reporter enhanced green fluorescence protein (EGFP) with empty vector (LVCO) or with OSK (LV-OSK). Confocal microscopy and western blotting analysis were used to confirm the OSK overexpression. Cellular migration, senescence, and apoptosis were evaluated. Human aortic ECs (HAoECs) from male and female normotensive and hypertensive patients were analyzed after OSK or control treatments for their endothelial nitric oxide synthase (eNOS) levels, nitric oxide (NO), and genetic profile. Male and female normotensive (BPN/3J) and hypertensive (BPH/2J) mice were treated with an intravenous (i.v.) injection of LVCO or LV-OSK and evaluated 10 days post-infection. The blood pressure, cardiac function, vascular reactivity of small arteries, in vivo EGFP signal and EndMT inhibition were analyzed. ResultsOSK overexpression induced partial EC reprogramming in vitro, and these cells showed endothelial progenitor cell (EPC)-like features with lower migratory capability. OSK treatment of hypertensive BPH/2J mice normalized blood pressure and resistance arteries hypercontractility, via the attenuation of EndMT and elastin breaks. EGFP signal was detected in vivo in the prefrontal cortex of both BPN/3J and BPH/2J-treated mice, but OSK induced angiogenesis only in male BPN/3J mice. OSK-treated human ECs from hypertensive patients showed high eNOS activation and NO production, with low ROS formation. Single-cell RNA analysis showed that OSK alleviated EC senescence and EndMT, restoring their phenotypes in human ECs from hypertensive patients. ConclusionOverall, these data indicate that OSK treatment and EC reprogramming can decrease blood pressure and reverse hypertension-induced vascular damage.

physiology↗

bioRxiv2024

Physics-driven structural docking and protein language models accelerate antibody screening and design for broad-spectrum antiviral therapy

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline AbGen that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. Our AbGen centers around a novel antibody language model (AbLM) that is pretrained on 12 million generic protein domain sequences and fine-tuned on 4,000+ paired VH-VL sequences, with IgG-specific CDR-masking and VH-VL cross-attention. AbLM provides a latent space of IgG sequence embeddings for AbGen, including (a) landscapes of IgGs activities in neutralizing the wild-type virus are analyzed through structure prediction for IgG and IgG-antigen (viral protein spikes receptor binding domain, RBD) interactions; and (b) landscapes of IgGs susceptibility in neutralizing variant viruses are predicted through Gaussian process regression, despite that as few as 14 clinical antibodies responses to variants of concern are available. The AbGen pipeline was applied to over 1300 IgG sequences we collected from RBD-binding B cells of convalescent patients. With experimental validations, AbGen efficiently prioritized IgG candidates against a broad spectrum of viral variants (wildtype, Delta, and Omicron), preventing the infection of host cells in vitro and hACE2 transgenic mice in vivo. Compared to other existing protein language models that require 10-100 times more model parameters, AbLM improved the precision from around 50% to 75% to predict IgGs with low variant susceptibility. Furthermore, AbGen enables structure-based computational protein redesign for selected IgG clones with single amino acid substitutions at the RBD-binding interface that doubled the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screen and re- design combining data-driven protein language models and Kriging for antibody sequence analysis and activity prediction, in synergy with physics-driven protein docking and design for antibody-antigen interface analyses and functional optimization.

immunology↗

bioRxiv2024

Blocker-SELEX: A Structure-guided Strategy for Developing Inhibitory Aptamers Disrupting Undruggable Transcription Factor Interactions

Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges associated with modulating their protein-protein and protein-DNA interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs do not favor the use of small-molecule inhibitors, or neutral antibodies, in blocking TF interactions. Aptamers are short oligonucleotides exhibiting high affinity and specificity for a diverse range of targets. Large molecular weights, expansive blocking surfaces and efficient cellular internalization make aptamers a compelling molecular tool for use as traditional TF interaction modulators. Here, we report a structure-guided design strategy called Blocker-SELEX to develop inhibitory aptamers (iAptamer) that selectively block TF interactions. Our approach led to the discovery of iAptamers that cooperatively disrupts SCAF4/SCAF8-RNA Polymerase II (RNAP2) interactions, thereby dysregulating RNAP2-dependent gene expression and splicing and, in turn, leading to the impairment of cell proliferation. This approach was further applied to develop iAptamers to efficiently block WDR5-MYC interaction with a nexus in cancer. Taken together, our study highlights the potential of Blocker-SELEX in developing iAptamers that effectively disrupt potentially pathogenic TF interactions with attendant implications for iAptamers as chemical tools for use in the study of biological functions of TF interactions, but also for potential use in nucleic acids drug discovery.

biochemistry↗