Biology subjects

Xu, S.

Publications and source records attributed to Xu, S..

At least 271 records · Page 15Linked to original sources

bioRxiv2023

Multiscale Model for Ion Transport in Cellular Media and Applications in Smooth Muscle Cells

Ion transport in biological tissues is crucial in the study of many biological and pathological problems. Some multi-cellular structures, like the smooth muscles on vessel walls, can be treated as periodic bi-domain structures consisting of the intracellular space (ICS) and extracellular space (ECS) with semipermeable membranes in between. In this work, we first use a multi-scale asymptotic method to derive a macroscopic homogenized bidomain model from the microscopic electro-neutral (EN) model with different diffusion coefficients and nonlinear interface conditions. Then, the obtained homogenized model is applied to study ion transportation and micro-circulation in multi-celluar tissues under the impact of agonists, an internal calcium source, and extracellular potassium. Our model serves as a useful bridge between existing ordinary differential equation models and partial differential models that take into consideration spatial variation. On the one hand, numerical results show that ECS variables are almost invariant in the first two scenarios and confirm the validity of existing single-domain models, which treat variables in the ECS as constants. On the other hand, only the bidomain model is applicable to consider the effect of local extracellular potassium. Finally, the membrane potential of syncytia formed by connected cells is found to play an important role in the propagation of oscillation from the stimulus region to the non-stimulus region. Author summarySmooth muscle cells (SMCs) play a vital role in neurovascular coupling, which is the mechanism by which changes in neural activity are linked to alterations in blood flow. Dysfunctional SMCs can have significant implications for human health. The activation of SMCs is primarily regulated by the intracellular concentration of calcium ions (Ca2+). A multi-scale model for ion transport in multicellular tissue with varying connectivity has been proposed to investigate SMC activation under different stimuli. The simulation results confirm the critical role of gap junctions in wave propagation and vasoconstriction in the vessel wall. The blockage of gap junctions prevents the spread of the wave. Furthermore, the propagation of membrane potential is the primary cause of wave propagation.

cell biology↗

bioRxiv2023

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 C atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.

biochemistry↗

bioRxiv2023

TREM2 mediates MHCII-associated CD4+ T cell response against gliomas

Triggering receptor expressed on myeloid cells 2 (TREM2) was recently highlighted as a novel immune suppressive marker in peripheral tumors. The aim of this study was to characterize TREM2 expression in gliomas and investigate its contribution in glioma progression by using Trem2-/-mouse line. Our results showed that higher TREM2 expression was correlated with poor prognosis in glioma patients. Unexpectedly, TREM2 deficiency did not have a beneficial effect in a pre-clinical model of glioma. The increased TREM2 expression in glioma was likely due to increased myeloid cell infiltration, as evidenced by our single-cell analysis showing that almost all microglia and macrophages in gliomas were TREM2+. Furthermore, we found that deficiency of TREM2 impaired tumor-myeloid phagocytosis and MHCII presentation, and significantly reduced CD4+ T cells in tumor hemispheres. Our results revealed a previously unrecognized protective role of tumor-myeloid TREM2 in promoting MHCII-associated CD4+ T cell response against gliomas. SUMMARYAuthors found that although higher TREM2 expression is correlated with poor prognosis in glioma patients, its absence has no beneficial effect in a pre-clinical model of glioma. Deficiency of TREM2 impairs myeloid cell phagocytosis of tumor debris, leading to a reduction in MHCII-dependent CD4+ anti-glioma immunity.

neuroscience↗

bioRxiv2023

PRMT1 Inhibition Selectively Targets BNC1-Dependent Proliferation, but not Migration in Squamous Cell Carcinoma

Squamous Cell Carcinoma (SCC) develops in stratified epithelial tissues and demonstrates frequent alterations in transcriptional regulators. We sought to discover SCC-specific transcriptional programs and identified the transcription factor Basonuclin 1 (BNC1) as highly expressed in SCC compared to other tumor types. RNA-seq and ChIP-seq analysis identified pro-proliferative genes activated by BNC1 in SCC cells and keratinocytes. Inhibition of BNC1 in SCC cells suppressed proliferation and increased migration via FRA1. In contrast, BNC1 reduction in keratinocytes caused differentiation, which was abrogated by IRF6 knockdown, leading to increased migration. Protein interactome analysis identified PRMT1 as a co-activator of BNC1-dependent proliferative genes. Inhibition of PRMT1 resulted in a dose-dependent reduction in SCC cell proliferation without increasing migration. Importantly, therapeutic inhibition of PRMT1 in SCC xenografts significantly reduced tumor size, resembling functional effects of BNC1 knockdown. Together, we identify BNC1-PRMT1 as an SCC-lineage specific transcriptional axis that promotes cancer growth, which can be therapeutically targeted to inhibit SCC tumorigenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/533164v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@97dd68org.highwire.dtl.DTLVardef@48ebaaorg.highwire.dtl.DTLVardef@167eb2corg.highwire.dtl.DTLVardef@180b325_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Standardization of suspension and imaging mass cytometry readouts for clinical decision making

Suspension and imaging mass cytometry are single-cell, proteomic-based methods used to characterize tissue composition and structure. Data assessing the consistency of these methods over an extended period of time are still sparse and are needed if mass cytometry-based methods are to be used clinically. Here, we present experimental and computational pipelines developed within the Tumor Profiler clinical study, an observational clinical trial assessing the relevance of cutting-edge technologies in guiding treatment decisions for advanced cancer patients. By using aliquots of frozen antibody panels, batch effects between independent experiments performed within a time frame of one year were minimized. The inclusion of well-characterized reference samples allowed us to assess and correct for batch effects. A systematic evaluation of a test tumor sample analyzed in each run showed that our batch correction approach consistently reduced signal variations. We provide an exemplary analysis of a representative patient sample including an overview of data provided to clinicians and potential treatment suggestions. This study demonstrates that standardized suspension and imaging mass cytometry measurements generate robust data that meet clinical requirements for reproducibility and provide oncologists with valuable insights on the biology of patient tumors.

cancer biology↗

bioRxiv2023

Body size as a metric to define objects in an affordable world

The physical body of an organism serves as a vital interface for interactions with its environment. Here we investigated the impact of human body size on the perception of action possibilities (affordances) offered by the environment. We found that the body size delineated a distinct boundary on affordances, dividing objects of continuous real-world sizes into two discrete categories with each affording distinct action sets. Additionally, the boundary shifted with imagined body sizes, suggesting a causal link between body size and affordance perception. Intriguingly, ChatGPT, a large language model lacking physical embodiment, exhibited a modest yet comparable affordance boundary at the scale of human body size, suggesting the boundary is not exclusively derived from organism-environment interactions. A subsequent fMRI experiment revealed evidence of affordance processing exclusively for objects within the body size range, but not for those beyond. This suggests that only objects capable of being manipulated are the objects capable of offering affordance in the eyes of an organism. In summary, our study suggests a novel definition of object-ness in an affordance-based context, advocating the concept of embodied cognition in understanding the emergence of intelligence constrained by an organisms physical attributes.

neuroscience↗

bioRxiv2023

Coupling of Slack and NaV1.6 sensitizes Slack to quinidine blockade and guides anti-seizure strategy development

Quinidine has been used as an anticonvulsant to treat patients with KCNT1-related epilepsy by targeting gain-of-function KCNT1 pathogenic mutant variants. However, the detailed mechanism underlying quinidines blockade against KCNT1 (Slack) remains elusive. Here, we report a functional and physical coupling of the voltage-gated sodium channel NaV1.6 and Slack. NaV1.6 binds to and highly sensitizes Slack to quinidine blockade. Homozygous knockout of NaV1.6 reduces the sensitivity of native sodium-activated potassium currents to quinidine blockade. NaV1.6-mediated sensitization requires the involvement of NaV1.6s N-and C-termini binding to Slacks C-terminus, and is enhanced by transient sodium influx through NaV1.6. Moreover, disrupting the Slack-NaV1.6 interaction by viral expression of Slacks C-terminus can protect against SlackG269S-induced seizures in mice. These insights about a Slack-NaV1.6 complex challenge the traditional view of "Slack as an isolated target" for anti-epileptic drug discovery efforts, and can guide the development of innovative therapeutic strategies for KCNT1-related epilepsy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/532982v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1127858org.highwire.dtl.DTLVardef@19ad9f4org.highwire.dtl.DTLVardef@15b5542org.highwire.dtl.DTLVardef@13a86b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

SAKit: an all-in-one analysis pipeline for identifying novel protein caused by variant events at genomic and transcriptic level

SummaryGenetic modifications that cause pivotal protein inactivation or abnormal activation may lead to cell signaling pathway change or even dysfunction, resulting in cancer and other diseases. In turn, dysfunction will further produce "novel proteins" that do not exist in the canonical human proteome. Identification of novel proteins is meaningful for identifying promising drug targets and developing new therapies. In recent years, several tools have been developed for identifying DNA or RNA variants with the extensive application of nucleotide sequencing technology. However, these tools mainly focus on point mutation and have limited performance in identifying large-scale variants as well as the integration of mutations. Here we developed a hybrid Sequencing Analysis bioinformatic pipeline by integrating all relevant detection Kits(SAKit): this pipeline fully integrates all variants at the genomic and transcriptomic level that may lead to the production of novel proteins defined as proteins with novel sequences compare to all reference sequences by comprehensively analyzing the long and short reads. The analysis results of SAKit demonstrate that large-scale mutations have more contribution to the production of novel proteins than point mutations, and long-read sequencing has more advantages in large-scale mutation detection. Availability and implementationSAKit is freely available on docker image (https://hub.docker.com/repository/docker/therarna/sakit), which is mainly implemented within a Snakemake framework in Python language.

bioinformatics↗

bioRxiv2023

The effects of mutations on gene expression and alternative splicing: a case study of EMS-induced heritable mutations in the microcrustacean Daphnia

Understanding the relationship between mutations and their genomic and phenotypic consequences has been a longstanding goal of evolutionary biology. However, few studies have investigated the impact of mutations on gene expression and alternative splicing on the genome- wide scale. In this study, we aim to bridge this knowledge gap by utilizing whole-genome sequencing data and RNA sequencing data from 16 OP Daphnia mutant lines to investigate the effects of EMS-induced mutations on gene expression and alternative splicing. Using rigorous analyses of mutations, expression changes, and alternative splicing, we show that trans-effects are the major contributor to the variance in gene expression and alternative splicing between the wildtype and mutant lines, whereas cis mutations only affected a limited number of genes and do not always alter gene expression. Moreover, we show that there is a significant association between DE genes and exonic mutations, indicating that exonic mutations are an important driver of altered gene expression.

evolutionary biology↗

bioRxiv2023

Comprehensive characterization of tumor microenvironment in colorectal cancer via histopathology-molecular analysis

PurposeTo explain how the tumor microenvironment (TME) contributes to biological and clinical heterogeneity of colorectal cancer (CRC). MethodsUsing multi-omics analysis, single cell transcriptomic sequencing analysis and artificial intelligence-enabled spatial analysis of whole-slide images, we performed a comprehensive characterization of TME in colorectal cancer (CCCRC). ResultsCRC samples were classified into four CCCRC subtypes with distinct TME features, namely, C1 as the proliferative subtype with low immunogenicity; C2 as the immunosuppressed subtype with the terminally exhausted immune characteristics; C3 as the immune-excluded subtype with the distinct upregulation of stromal components and a lack of T cell infiltration in tumor core; and C4 as the immunomodulatory subtype with the remarkable upregulation of anti-tumor immune components. The four CCCRC subtypes had distinct histopathological and molecular characteristics, therapeutic efficacy, and prognosis. The C1 subtype was more sensitive to chemotherapy, the C2 and C3 subtypes were more sensitive to WNT pathway inhibitor SB216763 and Hedgehog pathway inhibitor vismodegib, and the C4 subtype was suitable for ICB treatment. Finally, we established a single-sample gene classifier for identifying the CCCRC subtypes. ConclusionsOur integrative analyses ultimately established a holistic framework to thoroughly dissect the TME of CRC, and the CCCRC classification system with high biological interpretability might facilitate biomarker discoveries and clinical treatment decisions in the future.

cancer biology↗

bioRxiv2023

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 [A] cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

biochemistry↗

bioRxiv2023

Remodeling of the Ribosomal Quality Control and Protein Translation by a Viral Ubiquitin Deconjugase

The strategies adopted by viruses to reprogram the protein translation and quality control machineries to promote infection are poorly understood. Here, we discovered that the viral ubiquitin deconjugase (vDUB) encoded in the large tegument protein of Epstein- Barr virus (EBV) regulates ribosomal stress responses. The vDUB participates in protein complexes that include the ubiquitin ligases ZNF598 and LTN1 and the UFM1 ligase UFL1. Upon ribosomal stalling, the vDUB counteracts the ubiquitination of 40S ribosome subunits, inhibits the degradation of translation-stalled polypeptides by the proteasome, and prevents UFMylation of the 60S particle, which impairs the ER-phagy- dependent clearance of stalled products. Inhibition of the ribosome quality control activates a GCN2-dependent integrated stress response that decreases global protein translation while promoting the readthrough of stall-inducing mRNAs. The vDUB enhances viral mRNAs translation and virus release during productive infection, pointing to a pivotal role in cell reprogramming that enables virus production and underlies the pathogenesis of EBV-associated cancers and autoimmune diseases. GRAPHIC SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/526464v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c7421dorg.highwire.dtl.DTLVardef@c59b8dorg.highwire.dtl.DTLVardef@1676b4eorg.highwire.dtl.DTLVardef@b5e111_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A Systematic Survey of Reversibly Covalent Dipeptidyl Inhibitors of the SARS-CoV-2 Main Protease

SARS-CoV-2 is the coronavirus pathogen of the currently prevailing COVID-19 pandemic. It relies on its main protease (MPro) for replication and pathogenesis. MPro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as MPro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 reversibly covalent dipeptidyl MPro inhibitors and characterized them on in vitro enzymatic inhibition potency, structures of their complexes with MPro, cellular MPro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that MPro has a flexible S2 pocket that accommodates dipeptidyl inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as (S)-2-azaspiro[4,4]nonane-3-carboxylate and (S)-2-azaspiro[4,5]decane-3-carboxylate have optimal characteristics. One compound MPI60 containing a P2 (S)-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability and can be potentially advanced to further preclinical tests.

biochemistry↗

bioRxiv2023

Benchmarking Algorithms for Gene Set Scoring of Single-cell ATAC-seq Data

Gene set scoring (GSS) has been routinely conducted for gene expression analysis of bulk or single-cell RNA-seq data, which helps to decipher single-cell heterogeneity and cell-type-specific variability by incorporating prior knowledge from functional gene sets. Single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) is a powerful technique for interrogating single-cell chromatin-based gene regulation, and genes or gene sets with dynamic regulatory potentials can be regarded as cell-type specific markers as if in scRNA-seq. However, there are few GSS tools specifically designed for scATAC-seq, and the applicability and performance of RNA-seq GSS tools on scATAC-seq data remain to be investigated. We systematically benchmarked ten GSS tools, including four bulk RNA-seq tools, five single-cell RNA-seq (scRNA-seq) tools, and one scATAC-seq method. First, using matched scATAC-seq and scRNA-seq datasets, we find that the performance of GSS tools on scATAC-seq data is comparable to that on scRNA-seq, suggesting their applicability to scATAC-seq. Then the performance of different GSS tools were extensively evaluated using up to ten scATAC-seq datasets. Moreover, we evaluated the impact of gene activity conversion, dropout imputation, and gene set collections on the results of GSS. Results show that dropout imputation can significantly promote the performance of almost all GSS tools, while the impact of gene activity conversion methods or gene set collections on GSS performance is more GSS tool or dataset dependent. Finally, we provided practical guidelines for choosing appropriate pre-processing methods and GSS tools in different scenarios.

bioinformatics↗

bioRxiv2023

Phage-Assisted, Active Site-Directed Ligand Evolution with a Genetically Encoded Nε-Butyryl-ʟ-Lysine to Identify a Cellularly Potent and Selective Inhibitor for the ENL YEATS Domain as an Anti-Leukemia Agent

Eleven-nineteen leukemia protein (ENL) plays pivotal roles in the leukemogenesis. As a YEATS domain protein, ENL reads histone acylation marks and recruits key transcription factors to leukemic drivers such as HOXA9, MEIS1, and MYB and therefore promotes leukemia development. The histone-reading function of ENL has been proven essential in the onset and progression of several acute leukemias, suggesting a putative therapeutic window for ENL inhibition. In this study, we developed a phage-assisted, active site-directed ligand evolution (PADLE) approach for the identification of potent and selective ENL inhibitors, where N{varepsilon}-butyryl-O_SCPLOWLC_SCPLOW-lysine (BuK) that possesses known target-protein interactions with the ENL YEATS domain was genetically incorporated into a phage display library to serve as a warhead to direct displayed peptides to the active site of ENL YEATS for enrichment. Using this novel strategy in combination with structure-activity relationship that replaced BuK with other ncAAs for de novo {pi}-{pi}-{pi} stacking interactions with two aromatic residues in ENL YEATS, selective and potent ENL inhibitors with a Kd value as low as 2.0 nM were identified. One pentapeptide inhibitor tENL-S1f displayed selective inhibition of ENL over other YEATS domains as well as strong cellular target engagement and on-target effects in inhibiting leukemia cell growth and suppressing the expression of ENL target genes. As the first of its kind study, the current work opens a large avenue of research of using PADLE to develop selective and potent peptidyl inhibitors for a large variety of epigenetic reader proteins.

biochemistry↗

bioRxiv2023

Leafhopper transmits soybean stay-green associated virus to legume plants

A novel geminivirus, soybean stay-green associated virus (SoSGV), was previously shown to cause soybean delayed senescence and is associated with the incidences of soybean stay-green syndrome. The transmission methods of SoSGV were not yet understood. We captured insects belonging to 24 distinct species in a soybean field with the SoSGV outbreak and detected the presence of SoSGV only in leafhoppers and bean bugs (Riptortus pedestris). Caged feeding experiments using captured leafhoppers and bean bugs from soybean fields showed that leafhoppers, but not bean bugs, are vectors transmitting SoSGV. The common brown leafhopper (Orosius orientalis) is identified as the dominant leafhopper species and can establish colonies feeding on soybean plants in experimental conditions. An investigation of SoSGV defective DNA revealed that soybean genomic DNA could be inserted into the SoSGV genome, while sequences from wild soybean, red bean, and cowpea were also identified. We further showed that the common brown leafhopper could transmit SoSGV to wild soybean and red bean plants, emphasizing the vector role of the leafhopper in the transmission of SoSGV in the field.

microbiology↗

bioRxiv2022

Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the 2{delta}1 and {beta}1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the conformational change of VSDII in response to variation in membrane potential is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

biophysics↗

bioRxiv2022

An Amber-Encoding Helper Phage for More Efficient Phage Display of Noncanonical Amino Acids

In the past two decades, phage display has emerged as a powerful technique for the identification of antibodies and peptide ligands for therapeutic targets. Using the amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, we and others have shown that the chemical space in phage display can be significantly expanded for drug discovery. However, the use of amber codon in phages results in poor phage yields and requires tedious processes to enrich amber codon-containing (amber obligate) phage clones. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for rapid and continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by the insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The afforded novel helper phage allowed for a continuous amber codon enrichment strategy for two different phage display libraries and demonstrated a 100-fold increase in selectivity for packaging of library plasmids in comparison with original helper phage plasmids. To demonstrate the applicability of the system, CMa13ile40 was used to create two phage-displayed peptide libraries containing two separate ncAAs, N{varepsilon} -tert-butoxycarbonyl-lysine (BocK) and N{varepsilon} -allyloxycarbonyl-lysine (AllocK), respectively. These were then used to identify peptide ligands that bind to the extracellular domain of ZNRF3, a membrane-bound E3 ligase. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Using biolayer interferometry, enriched peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 and this affinity is dependent on the presence of the ncAA used for selection. Our results clearly show that ncAAs in phages provide unique interactions for selection of peptides that are different from each other and from canonical amino acids. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

biochemistry↗