The MLLE domain is a peptide-binding domain found in the poly(A) binding protein (PABP) and the ubiquitin protein E3 ligase N-recognin 5 (UBR5) that recognizes a conserved motif, named PABP-interacting motif 2 (PAM2). The majority of PAM2 sequences bind to MLLE domains with low-micromolar affinity. Here, we designed a chimeric PAM2 peptide termed super PAM2 (sPAM2) by combining classical and trinucleotide repeat-containing 6 (TNRC6)-like binding modes to create a superior binder for the MLLE domain. The crystal structure of the PABPC1 MLLE-sPAM2 complex shows a crucial role of conserved sPAM2 leucine, phenylalanine and tryptophan residues in the interaction. We used deep mutational scanning (DMS) coupled with isothermal titration calorimetry (ITC) to characterize the specificity profiles for PABPC1 and UBR5 MLLE. The best sPAM2 sequence binds to PABPC1 MLLE with low-nanomolar affinity and nearly 20-fold more tightly than the best natural PAM2 sequence. This suggests that the affinities of natural PAM2 sequences are tuned to control their binding to PABPC1 and UBR5. Our study will aid in the discovery of new PAM2-containing proteins (PACs) and facilitate in vivo studies of PAM2-mediated cellular pathways.

Salmonella contamination in food and water poses a major global health risk, creating an urgent need for rapid, reliable, and cost-effective detection methods. Conventional approaches are often expensive, labor-intensive, and time-consuming, and they frequently yield inconclusive or presumptive positive results. A significant bottleneck to rapid detection is the need to separate the target bacteria into smaller, clean, and concentrated samples. Bacteriophages can recognize, bind, and infect specific bacterial hosts. This study presents a genetically engineered S16 Salmonella-specific bacteriophage as a biosensor, optimized for enhanced sensitivity and efficiency in detecting Salmonella. Using CRISPR-Cas12a, the phages were engineered to include a gene for a NanoLuc luciferase reporter and a monomeric streptavidin (mSA) affinity tag fused to the gene for the capsid protein Soc. This design enabled conjugation of the phages to magnetic nanoparticles, facilitating the capture, concentration, and detection of Salmonella from 10 mL water samples. The modified S16 phage exhibited a detection limit of fewer than 10 CFU of Salmonella in 10 mL of water within a typical work shift. This innovative phage-based detection method offers a promising tool for enhancing food and water safety by providing a faster, more sensitive, and cost-effective approach to pathogen monitoring of Salmonella enterica subsp. enterica serovar Typhimurium.

Bovine rotavirus (BRV) is one of the main pathogens that cause acute diarrhea in calves under one month of age. Passive immunization has been recognized as an effective way to prevent and treat BRV infection. Recent studies have shown that 10% of bovine antibodies possess an ultra-long CDR H3 domain, which has been shown to be the smallest antigen-binding domain. Due to the extremely small size of ultra-long CDR H3 antibodies, the phage display method was utilized to obtain ultra-long CDR H3 antibodies targeting BRV, providing a new approach for the prevention and/or treatment of BRV. Here, we report the preparation of BRV-specific bovine ultra-long CDR H3 antibodies obtained by constructing and screening a phage display library containing approximately 8.55 × 109 individual clones. Through three rounds of bio-panning, we identified 92 candidate clones, of which 79 exhibited specific binding activity in phage ELISAs. The recombinant bovine ultra-long CDR H3 antibodies could specifically bind to BRV in ELISAs and cell immunofluorescence assays. The neutralizing activity was further confirmed through virus neutralization tests. In the calf model experiment, the recombinant bovine ultra-long CDR H3 antibodies could relieve the symptoms of diarrhea, reduce both the amount and duration of virus release, and increase the survival in calves experimentally infected with BRV. Therefore, BRV-specific bovine ultra-long CDR H3 antibodies could serve as an effective agent for the prevention and treatment of BRV infection. At the same time, the development of ultra-long CDR H3 antibodies using phage display screening technology provides a new approach for developing biological agents for the prevention and control of infectious diseases in bovines.

Peptides are bioactive intermediates between small organic molecules and large biological compounds like antibodies or proteins. These compounds play a unique and valuable role as therapeutic agents, owing to their unique biochemical properties and versatility in treating a wide range of diseases such as metabolic disorders, cancer therapy, antimicrobial and anti-inflammatory agents. The global peptide therapeutics market is projected to exceed USD 50 billion by 2024, reflecting the increasing demand and interest in this field. Therapeutic peptides offer an optimal balance of specificity, safety, and molecular size, providing greater precision in targeting specific receptors with fewer off-target effects and reduced toxicity compared to small-organic drugs. Peptides also exhibit enhanced tissue penetration and present simpler, cheaper manufacturing processes with lower immunogenicity. To date, around 100 peptides have attained clinical approval in major markets, with nearly half of these approvals occurring in the past 20 years. This trend highlights the growing importance and therapeutic potential of peptides in modern medicine, explaining the substantial market associated with these treatments. The review presents a detailed comparison of the major parenteral administration modes for therapeutic peptides, specifically subcutaneous and intravenous routes. We highlight how these methods impact the pharmacokinetic profiles of peptides and influence patient outcomes, providing critical insights into the advantages and limitations of each route. Finally, a significant aspect of this review is its focus on innovative drug delivery systems and formulations designed to address the challenges of peptide delivery, namely stability, bioavailability, and therapeutic efficacy.

Bacteriophages, with their distinctive ability to selectively target host bacteria, stand out as a compelling tool in the realm of drug and gene delivery. Their assembly from proteins and nucleic acids, coupled with their modifiable and biologically unique properties, enables them to serve as efficient and safe delivery systems. Unlike conventional nanocarriers, which face limitations such as non-specific targeting, cytotoxicity, and reduced transfection efficiency in vivo, engineered phages exhibit promising potential to overcome these hurdles and improve delivery outcomes. This review highlights the potential of bacteriophage-based systems as innovative and efficient systems for delivering therapeutic agents. It explores strategies for engineering bacteriophage, categorizes the principal types of phages employed for drug and gene delivery, and evaluates their applications in disease therapy. It provides intriguing details of the use of natural and engineered phages in the therapy of diseases such as cancer, bacterial and viral infections, veterinary diseases, and neurological disorders, as well as the use of phage display technology in generating monoclonal antibodies against various human diseases. Additionally, the use of CRISPR-Cas9 technology in generating genetically engineered phages is elucidated. Furthermore, it provides a critical analysis of the challenges and limitations associated with phage-based delivery systems, offering insights for overcoming these obstacles. By showcasing the advancements in phage engineering and their integration into nanotechnology, this study underscores the potential of bacteriophage-based delivery systems to revolutionize therapeutic approaches and inspire future innovations in medicine.

Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein-protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein-protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 spike-ACE2 protein-protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein-protein interactions with long lasting effects.

The nascent field of targeted protein degradation (TPD) could revolutionize biomedicine due to the ability of degrader molecules to selectively modulate disease-relevant proteins. A key limitation to the broad application of TPD is its dependence on small-molecule ligands to target proteins of interest. This leaves unstructured proteins or those lacking defined cavities for small-molecule binding out of the scope of many TPD technologies. The use of proteins, peptides, and nucleic acids (otherwise known as "biologics") as the protein-targeting moieties in degraders addresses this limitation. In the following sections, we provide a comprehensive and critical review of studies that have used proteins and peptides to mediate the degradation and hence the functional control of otherwise challenging disease-relevant protein targets. We describe existing platforms for protein/peptide-based ligand identification and the drug delivery systems that might be exploited for the delivery of biologic-based degraders. Throughout the Review, we underscore the successes, challenges, and opportunities of using protein-based degraders as chemical biology tools to spur discoveries, elucidate mechanisms, and act as a new therapeutic modality.

Affinity selection-mass spectrometry (AS-MS) is a ligand discovery platform that relies upon mass spectrometry to identify molecules bound to a biomolecular target. When utilized with large peptide libraries (108 members), AS-MS sample complexity can surpass the sequencing capacity of modern mass spectrometers, resulting in incomplete data, identification of few target-specific ligands, and/or incomplete sequencing. To address this challenge, we introduce pyBinder to perform quantitation on AS-MS data to process primary MS1 data and develop two scores to rank the peptides from the integration of their peak area: target selectivity and concentration-dependent enrichment. We benchmark pyBinder utilizing AS-MS data developed against antihemagglutinin antibody 12ca5, revealing that peptides that contain a motif known for target-specific high-affinity binding are well characterized by these two scores. AS-MS data from a second protein target, WD Repeat Domain 5 (WDR5), is analyzed to confirm the two pyBinder scores reliably capture the target-specific motif-containing peptides. From the results delivered by pyBinder, a list of target-selective features is developed and fed back into subsequent MS experiments to facilitate expanded data generation and the targeted discovery of selective ligands. pyBinder analysis resulted in a 4-fold increase in motif-containing sequence identification for WDR5 (from 3 to 14 ligands discovered), showing the utility of the two scores. This work establishes an improved approach for AS-MS to enable discovery outcomes (i.e., more ligands identified), but also a way to compare AS-MS data across samples, protocols, and conditions broadly.

Aptamer technology has significantly advanced the field of plant research, emerging as a tool for enhancing agricultural productivity, plant growth, and environmental monitoring. Aptamers are short nucleotide or amino acid sequences that can bind to a range of target molecules with high affinity and selectivity. In recent years, these affinity molecules have piqued the interest of researchers across various scientific fields, including pharmaceuticals, analytical chemistry, and plant science. Advancements in aptamer technology have significantly broadened the horizons of plant science, particularly in the areas of plant analyte detection, pathogen targeting, and protein function analysis. Despite the use of various other bioassays and molecular techniques for plant analyte detection, the small size, chemical stability, and cost-effective synthesis of aptamers make them invaluable tools for unravelling the complexities of plant cells. Here, we discuss the progress in the development of nucleic acid and peptide aptamers and summarize their applications in plant biotechnology. The principles and signalling methods of various aptamer-based biosensors and their prospects as biotechnological tools for functional genomic studies, pathogen resistance, and bioimaging are discussed. Finally, the present challenges and future perspectives of aptamer-based technology in plant research are also summarized.

Antibodies are naturally produced safeguarding proteins that the immune system generates to fight against invasive invaders. For centuries, they have been produced artificially and utilized to eradicate various infectious diseases. Given the ongoing threat posed by COVID-19 pandemics worldwide, antibodies have become one of the most promising treatments to prevent infection and save millions of lives. Currently, in silico techniques provide an innovative approach for developing antibodies, which significantly impacts the formulation of antibodies. These techniques develop antibodies with great specificity and potency against diseases such as SARS-CoV-2 by using computational tools and algorithms. Conventional methods for designing and developing antibodies are frequently costly and time-consuming. However, in silico approach offers a contemporary, effective, and economical paradigm for creating next-generation antibodies, especially in accordance with recent developments in bioinformatics. By utilizing multiple antibody databases and high-throughput approaches, a unique antibody construct can be designed in silico, facilitating accurate, reliable, and secure antibody development for human use. Compared to their traditionally developed equivalents, a large number of in silico-designed antibodies have advanced swiftly to clinical trials and became accessible sooner. This article helps researchers develop SARS-CoV-2 antibodies more quickly and affordably by giving them access to current information on computational approaches for antibody creation.

To develop efficient selection strategies and improve the discovery of promising ligands, it is highly desirable to analyze the sequence composition of naïve phage display libraries and monitor the evolution of their peptide content during successive rounds of amplification. In the current study, we performed a comparative analysis of the compositional features in different lots of the same naïve phage display library and monitored alterations in their peptide compositions during three rounds of amplification.

We conducted three rounds of duplicate serial amplification of two different lots of the Ph.D.™-12 phage display library. DNA from the samples was subjected to Next-Generation Sequencing (NGS) using an Illumina platform. The NGS datasets underwent a variety of bioinformatic analyses using Python and MATLAB scripts.

We observed substantial heterogeneity in the sequence composition of the two lots indicated by differences in the enhanced percentage of wildtype clones, reduced diversity (number of unique sequences), and increased enrichment factors (EFs) during amplification as well as by observing no common sequence between lots and decreased number of common sequences between the naïve library and the consecutive rounds of amplification for each lot. We also found potential propagation-related target-unrelated peptides (TUPs) with the highest EFs in the two lots, which were displayed by the fastest-propagating phage clones. Furthermore, motif analysis of the most enriched subpopulation of amplified libraries led to the identification of some motifs hypothesized to contribute to the increased amplification rates of the respective phage clones.

Our results highlight tremendous heterogeneity in the peptide composition of different lots of the same type of naïve phage display library, and the divergent evolution of their compositional features during amplification rounds at the amino acid, peptide, and motif levels. Our findings can be instrumental for phage display researchers by bringing fundamental insights into the vast extent of non-uniformity between phage display libraries and by providing a clear picture of how these discrepancies can lead to different evolutionary fates for the peptide composition of phage pools, which can have profound impacts on the outcome of phage display selections through biopanning.

The number of metal-containing waste streams resulting from electronic end-of life products, metallurgical by-products, and mine tailings to name but a few, is increasing worldwide. In recent decades, the potential to exploit these waste streams as valuable secondary resources to meet the high demand of critical and economically important raw materials has become more prominent. In this review, fundamental principles of bio-based metal recovery technologies are discussed focusing on microbial metabolism-dependent and metabolism-independent mechanisms as sustainable alternatives to conventional chemical metal recovery methods. In contrast to previous reviews which have partially addressed this topic, a special focus will be given on how fundamental principles of bio-based recovery technologies can influence the selectivity and specificity of metal recovery. While conventional methods for metal recovery show benefits in terms of economic affordability, bio-based recovery technologies offer advantages in terms of efficiency and environmentally friendliness. Modifications and adaptations in the processes of biosorption, bioaccumulation and bioelectrochemical systems are highlighted, further emphasizing the application of metal-binding peptides and siderophores to increase selectivity in the recovery of metals. Single metal solutions or mixtures with a low complexity have been the focus of previous studies and reviews, but this does not reflect the nature of complex industrial effluents. Therefore, key challenges that arise when dealing with complex polymetallic solutions are addressed and the focus is set on optimizing bio-based technologies to recover metals efficiently and selectively from bio-leachates or liquid waste streams.

Antibodies and antibody mimics are extensively used in the pharmaceutical industry, where stringent safety standards are required. Implementing heat sterilization during or after the manufacturing process could help prevent contamination by viruses and bacteria. However, conventional antibodies and antibody mimics are not suitable for heat sterilization because they irreversibly denature at high temperatures. In this study, we focused on the refolding property of the cold shock protein from the hyperthermophile Thermotoga maritima (TmCSP), which denatures at elevated temperatures but regains its native structure upon re-cooling. We designed and constructed a mutant library of TmCSP in which amino acid residues in its three surface loops were diversified. From the library, mutant TmCSPs that bind to each of eight target proteins were selected by phage and yeast surface display methods. We confirmed that the secondary structure and binding affinity of all the selected mutants were restored after heat treatment followed by cooling. Additionally, freeze-drying did not impair their binding affinity. The crystal structure of a mutant TmCSP in complex with its target, the esterase from Alicyclobacillus acidocaldarius, revealed specific interactions between them. These results clearly demonstrate the feasibility of creating heat-sterilizable antibody mimics using TmCSP as a scaffold.

The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.

Computational generation of cyclic peptide inhibitors using machine learning models requires large size training data sets often difficult to generate experimentally. Here we demonstrated that sequential combination of Random Forest Regression with the pseudolikelihood maximization Direct Coupling Analysis method and Monte Carlo simulation can effectively enhance the design pipeline of cyclic peptide inhibitors of a tumor-associated protease even for small experimental data sets. Further in vitro studies showed that such in silico-evolved cyclic peptides are more potent than the best peptide inhibitors previously developed to this target. Crystal structure of the cyclic peptides in complex with the protease resembled those of protein complexes, with large interaction surfaces, constrained peptide backbones, and multiple inter- and intramolecular interactions, leading to good binding affinity and selectivity.

Selective control over crystallization in complex multicomponent systems such as hydrating cements is a key issue in modern material science. In this context, rational selection-based approaches appear highly promising in the quest for new additive chemistries. Here we have used phage display to identify peptide structures showing high affinity to adsorb on the surfaces of calcium sulfate hemihydrate (also referred to as bassanite), an important hydraulic binder employed in large scales by the construction industry. The results suggest a triplet of amino acids consisting of aspartic acid, serine and leucine, to maintain strong interactions with the surfaces of hemihydrate particles. This notion is confirmed by actual hydration experiments, in which the identified peptide motif provides strictly selective effects during the transformation of bassanite into more stable gypsum. Our work thus contributes to a better understanding of hydraulic binders and their required improvement for a sustainable future.

Background: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. RiPPs have attracted attention for the ability to generate and screen libraries of these compounds for useful biological activities. To facilitate this screening, it is useful to be able to do so with the leader peptide still present. We assessed the suitability of the microviridin family for these screening experiments by determining their activity with the leader peptide still present. Methods: Modified precursor peptides with the leader present were heterologously expressed in Escherichia coli. Their ability to inhibit elastase was tested with a fluorogenic substrate. HPLC was used to monitor degradation of the modified precursor peptides by elastase. SDS-PAGE was used to determine the ability of immobilized modified precursor peptide to pull down elastase. Results: We found that the fully modified precursor peptide of microviridin B can inhibit the serine protease elastase with a low nanomolar IC50, and that the fully modified precursor with an N-terminal His-tag can mediate interactions between elastase and Ni-NTA resin, all indicating leader peptide removal is not necessary for microviridins to bind their target proteases. Additionally, we found that a bicyclic variant was able to inhibit elastase with the leader peptide still present, although with a roughly 100-fold higher IC50 and being subject to hydrolysis by elastase. Conclusions: These results open a pathway to screening libraries of microviridin variants for improved protease inhibition or other characteristics that can serve as, or as inspirations for, new pharmaceuticals.

For cystic fibrosis patients, a lung-targeted gene therapy would significantly alleviate pulmonary complications associated with morbidity and mortality. However, mucus in the airways and cell entry pose huge delivery barriers for local gene therapy. Here, we used phage display technology to select for and identify mucus- and cell-penetrating peptides against primary human bronchial epithelial cells from cystic fibrosis patients cultured at the air-liquid interface. At the air-liquid interface, primary human bronchial epithelial cells produce mucus and reflect cystic fibrosis disease pathology, making it a clinically relevant model. Using this model, we discovered a lead candidate peptide and incorporated it into lipid nanoparticles to deliver mRNA to primary human bronchial epithelia in vitro and mouse lungs in vivo. Compared to lipid nanoparticles without our peptide, peptide-lipid nanoparticles demonstrated up to 7.8-fold and 3.4-fold higher reporter luciferase bioactivity in vitro and in vivo, respectively. Importantly, these peptides facilitated higher specific uptake of nanoparticles into lung epithelia relative to other cell types. Since gene delivery to primary human bronchial epithelia is a significant challenge, we are encouraged by these results and anticipate that our peptide could be used to successfully deliver cystic fibrosis gene therapies in future work.

Novel bioconjugation reactions have been heavily pursued for the past two decades. A myriad of conjugation reactions have been developed for labeling molecules of interest in their native context as well as for constructing multifunctional molecular entities or stimuli-responsive materials. A growing cluster of bioconjugation reactions were realized by tapping into the unique properties of boron. As a rare element in human biology, boronic acids and esters exhibit remarkable biocompatibility. A number of organoboron reagents have been evaluated for bioconjugation, targeting the reactivity of either native biomolecules or those incorporating bioorthogonal functional groups. Owing to the dynamic nature of B-O and B-N bond formation, a significant portion of the boron-enabled bioconjugations exhibit rapid reversibility and accordingly have found applications in the development of reversible covalent inhibitors. On the other hand, stable bioconjugations have been developed that display fast kinetics and significantly expand the repertoire of bioorthogonal chemistry. This contribution presents a summary and comparative analysis of the recently developed boron-mediated bioconjugations. Importantly, this article seeks to provide an in-depth discussion of the thermodynamic and kinetic profiles of these boron-enabled bioconjugations, which reveals structure-reactivity relationships and provides guidelines for bioapplications.

Electrochemical biosensing continues to advance tirelessly, overcoming barriers that have kept it from leaving research laboratories for many years. Among them, its compromised performance in complex biological matrices due to fouling or receptor stability issues, the limitations in determining toxic and small analytes, and its use, conditioned to the commercial availability of commercial receptors and the exploration of natural molecular interactions, deserved to be highlighted. To address these challenges, in addition to the intrinsic properties of electrochemical biosensing, its coupling with biomimetic materials has played a fundamental role, among which bioinspired phage and peptide probes stand out. The versatility in design and employment of these probes has opened an unimaginable plethora of possibilities for electrochemical biosensing, improving their performance far beyond the development of highly sensitive and selective devices. The state of the art offers robust electroanalytical biotools, capable of operating in complex samples and with exciting opportunities to discover and determine targets regardless of their toxicity and size, the commercial availability of bioreceptors, and prior knowledge of molecular interactions. With all this in mind, this review offers a panoramic, novel, and updated vision of both the tremendous advances and opportunities offered by the combination of electrochemical biosensors with bioinspired phage and peptide probes and the challenges and research efforts that are envisioned in the immediate future.

Peptides are important biomarkers for various diseases, however distinguishing specific amino-acid sequences using artificial receptors remains a major challenge in biomedical sensing. This study introduces a new approach for creating highly selective recognition surfaces using phage display biopanning against metal-organic nanosheets (MONs). Three MONs (ZIF-7, ZIF-7-NH2, and Hf-BTB-NH2) are added to a solution containing every possible combination of seven-residue peptides attached to bacteriophage hosts. The highest affinity peptides for each MON are isolated through successive bio-panning rounds. Comparison of the surface properties of the MONs and high-affinity peptides provide useful insights into the relative importance of electrostatic, hydrophobic, and co-ordination bonding interactions in each system, aiding the design of future MONs. Coating of the Hf-BTB-NH2 MONs onto a quartz crystal microbalance (QCM) produced a five-fold higher signal for phage with the on-target peptide sequence compared to those with generic sequences. Surface plasmon resonance (SPR) studies produce a 4600-fold higher equilibrium dissociation constant (KD) for on-target sequences and are comparable to those of antibodies (KD = 4 x 10-10 m). It is anticipated that insights from the biopanning approach, combined with the highly tunable nature of MONs, will lead to a new generation of highly selective recognition surfaces for use in biomedical sensors.

Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein-protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein-protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 Spike-ACE2 protein-protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein-protein interactions with long lasting effects.

Phage display is a vital tool for the discovery and development of affinity reagents such as antibodies and peptides, which have great potential in imaging, molecular recognition, biosensors, targeted delivery and other clinical applications. However, affinity reagents obtained by phage display are often subjected to a process called biopanning, which is considered time-consuming, labor-intensive and lacks accurate control, limiting the acquisition of high-quality affinity reagents. Over the last two decades, several microfluidic approaches have been designed to simplify the conventional biopanning process and to realize precise control. To better understand the advantages of microfluidics over traditional biopanning and the potential of microfluidics for other molecular screening strategies, we provided an overview of recent applications of microfluidics in phage display. Additionally, the next challenges and outlooks are discussed.

A key factor in biomineralization is the use of organic molecules to direct the formation of inorganic materials. However, identification of molecules that can selectively produce the calcium carbonate polymorphs calcite or aragonite has proven extremely challenging. Here, we use a phage display approach to identify proteins - rather than the short peptides typically identified using this method - that can direct calcium carbonate formation. A 1.3 × 1010 library of Affimer proteins was displayed on modified M13 phage, where an Affimer is a ≈13 kDa protein scaffold that displays two variable regions of 9-13 residues. The phage displaying the Affimer library were then screened in binding assays against calcite and aragonite at pH 7.4, and four different strongly-binding proteins were identified. The two aragonite-binding proteins generated aragonite when calcium and magnesium ions were present at a 1 : 1 ratio, while the calcite-binding proteins produce magnesium-calcite under the same conditions. Calcite alone formed in the presence of all four proteins in the absence of magnesium ions. In combination with molecular dynamics simulations to evaluate the conformations of the proteins in solution, this work demonstrates the importance of conformation in polymorph control, and highlights the importance of magnesium ions, which are abundant in seawater, to reduce the energetic barriers associated with aragonite formation.

Despite considerable progress in using lipid nanoparticle (LNP) vehicles for gene delivery, achieving selective transfection of specific cell types remains a significant challenge, hindering the advancement of new gene or gene-editing therapies. Although LNPs have been equipped with ligands aimed at targeting specific cellular receptors, achieving complete selectivity continues to be elusive. The exact reasons for this limited selectivity are not fully understood, as cell targeting involves a complex interplay of various cellular factors. Assessing how much ligand/receptor binding contributes to selectivity is challenging due to these additional influencing factors. Nonetheless, such data are important for developing new nanocarriers and setting realistic expectations for selectivity. Here, we have quantified the selective, targeted transfection using two uniquely engineered cell lines that eliminate unpredictable and interfering cellular influences. We have compared the targeted transfection of Chinese ovary hamster (CHO) cells engineered to express the human transferrin receptor 1 (hTfR1), CHO-TRVb-hTfR1, with CHO cells that completely lack any transferrin receptor, CHO-TRVb-neo cells (negative control). Thus, the two cell lines differ only in the presence/absence of hTfR1. The transfection was performed with pDNA-encapsulating LNPs equipped with the DT7 peptide ligand that specifically binds to hTfR1 and enables targeted transfection. The LNP's pDNA encoded for the monomeric GreenLantern (mGL) reporter protein, whose fluorescence was used to quantify transfection. We report a novel LNP composition designed to achieve an optimal particle size and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting capability, and sufficient polyethylene glycol sheltering to minimize random cell targeting. The transfection efficiency was quantified in both cell lines separately through flow cytometry based on the expression of the fluorescent gene product. Our results demonstrated an LNP dose-dependent mGL expression, with a 5-fold preference for the CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment, when both cell lines were mixed at a 1:1 ratio, the DT7-decorated LNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over the CHO-TRVb-neo cells. Based on the low-level transfection of the CHO-TRVb-neo cells in both experiments, our results suggest that 17-25% of the transfection occurred in a nonspecific manner. The observed transfection selectivity for the CHO-TRVb-hTfR1 cells was based entirely on the hTfR1/DT7 interaction. This work showed that the platform of two engineered cell lines which differ only in the hTfR1 can greatly facilitate the development of LNPs with hTfR1-targeting ligands.

The development of robust NMR methodologies to probe dynamics on the atomic scale is vital to elucidate the close relations between structure, motion, and function in biological systems. Here, we present an automated protocol to measure, using magic-angle spinning NMR, the effective 13C-15N dipolar coupling constants between multiple spin pairs simultaneously with high accuracy. We use the experimental dipolar coupling constants to quantify the order parameters of multiple C-N bonds in the thousands of identical copies of the coat protein in intact fd-Y21M filamentous bacteriophage virus and describe its overall dynamics on the submillisecond time scale. The method is based on combining three pseudo three-dimensional NMR experiments, where a rotational echo double resonance (REDOR) dephasing block, designed to measure internuclear distances, is combined with three complementary 13C-13C mixing schemes: dipolar-assisted rotational resonance, through-bond transfer-based double quantum/single quantum correlation, and radio frequency driven recoupling. These mixing schemes result in highly resolved carbon spectra with correlations that are created by different transfer mechanisms. We show that the helical part of the coat protein undergoes a uniform small (∼30°) amplitude motion, while the N-terminus is highly flexible. In addition, our results suggest that the reduced mobility of lysine sidechains at the C-terminus are a signature of binding to the single stranded DNA.

Phage display has been widely used to identify peptides binding to a variety of biological targets. In the current work, we planned to select novel peptides targeting CD4 through screening of a commercial phage display library (New England Biolabs Ph.D.TM-7). After three rounds of biopanning, 57 phage clones were Sanger-sequenced. These clones represented 30 unique peptide sequences, which were subjected to phage ELISA, resulting in the identification of two potential target binders. Following peptide synthesis, downstream characterization was conducted using fluorescence plate-based assay, flow cytometry, SPR, and confocal microscopy. The results revealed that neither of the peptides identified in the Sanger-based phage display selection exhibited specific binding toward CD4. The naïve library and the phage pool recovered from the third round of biopanning were then subjected to next-generation sequencing (NGS). The results of NGS indicated corruption of the selection output by a phage already known as a fast-propagating clone whose target-unrelated enrichment can shed light on the misidentification of target-binding peptides through phage display. This work provides an in-depth insight into some of the challenges encountered in peptide phage display selection. Furthermore, our data highlight that NGS, by exploring a broader sequence space and providing a more precise picture of the composition of biopanning output, can be used to refine the selection protocol and avoid misleading the process of ligand identification. We hope that these findings can describe some of the complexities of phage display selection and offer help to fellow researchers who have faced similar situations.

Calcium phosphates (CaPs) and their substituted derivatives encompass a large number of compounds with a vast presence in nature that have aroused a great interest for decades. In particular, hydroxyapatite (HAp, Ca10(OH)2(PO4)6) is the most abundant CaP mineral and is significant in the biological world, at least in part due to being a major compound in bones and teeth. HAp exhibits excellent properties, such as safety, stability, hardness, biocompatibility, and osteoconductivity, among others. Even some of its drawbacks, such as its fragility, can be redirected thanks to another essential feature: its great versatility. This is based on the compound's tendency to undergo substitutions of its constituent ions and to incorporate or anchor new molecules on its surface and pores. Thus, its affinity for biomolecules makes it an optimal compound for multiple applications, mainly, but not only, in biological and biomedical fields. The present review provides a chemical and structural context to explain the affinity of HAp for biomolecules such as proteins and nucleic acids to generate hybrid materials. A size-dependent criterium of increasing complexity is applied, ranging from amino acids/nucleobases to the corresponding macromolecules. The incorporation of metal ions or metal complexes into these functionalized compounds is also discussed.

While monoclonal antibodies have shown success in cancer immunotherapy, their limitations prompt exploration of alternative approaches such as aptamers and peptides targeting programmed death ligand 1 (PD-L1). Despite the significance of these biotechnological tools, a comprehensive review encompassing both aptamers and peptides for PD-L1 targeting is lacking. Addressing this gap is crucial for consolidating recent advancements and insights in this field. Biotechnological advances leveraging aptamers and peptides represent a cutting-edge approach in refining the targeting proteins. Our review aims to provide valuable guidance for researchers and clinicians, highlighting the biotechnological advances utilizing aptamers and peptides refining PD-L1 targeting.

Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques.

Chirality is ubiquitous in nature across all length scales, with major implications spanning fields from biology, chemistry and physics to materials science. How chirality propagates from nanoscale building blocks to meso- and macroscopic helical structures remains an open issue. Here, working with a canonical system of filamentous viruses, we demonstrate that their self-assembly into chiral liquid crystal phases quantitatively results from the interplay between two main mechanisms of chirality transfer: electrostatic interactions from the helical charge patterns on the virus surface, and fluctuation-based helical deformations leading to viral backbone helicity. Our experimental and theoretical approach provides a comprehensive framework for deciphering how chirality is hierarchically and quantitatively propagated across spatial scales. Our work highlights the ways in which supramolecular helicity may arise from subtle chiral contributions of opposite handedness that act either cooperatively or competitively, thus accounting for the multiplicity of chiral behaviours observed for nearly identical molecular systems.

Biomaterials in nature form hierarchical structures and functions across various length scales through binding and assembly processes. Inspired by nature, we developed hierarchically organized tissue engineering materials through evolutionary screening and self-templating assembly. Leveraging the M13 bacteriophage (phage), we employed an evolutionary selection process against hydroxyapatite (HA) to isolate HA-binding phage (HAPh). The newly discovered phage exhibits a bimodal length, comprising 950 nm and 240 nm, where the synergistic effect of these dual lengths promotes the formation of supramolecular fibrils with periodic banded structures. The assembled HAPh fibrils show the capability of HA mineralization and the directional growth of osteoblast cells. When applied to a dentin surface, it induces the regeneration of dentin-like tissue structures, showcasing its potential applications as a scaffold in tissue engineering. The integration of evolutionary screening and self-templating assembly holds promise for the future development of hierarchically organized tissue engineering materials.

Gold nanoparticles (AuNPs), because of their dual plasmonic and catalytic functionalities, are among the most promising nanomaterials for the development of therapeutic and diagnostic tools for severe diseases such as cancer and neurodegeneration. Bacteriophages, massively present in human biofluids, are emerging as revolutionary biotechnological tools as they can be engineered to display multiple specific binding moieties, providing effective targeting ability, high stability, low cost, and sustainable production. Coupling AuNPs with phages can lead to an advanced generation of nanotools with great potential for biomedical applications. In the present study, we analyzed the interactions between differently sized AuNPs and filamentous M13 phages, establishing an advanced characterization platform that combines analytical techniques and computational models for an in-depth understanding of these hybrid self-assembling systems. A precise and structurally specific interaction of the AuNP-M13 hybrid complexes was observed, leading to a peculiar head/tail "tadpole-like" configuration. In silico simulations allowed explaining the mechanisms underlying the preferential assembly route and providing information about AuNPs' size-dependent interplay with specific M13 capsid proteins. The AuNP-M13 structures were proven to be biomimetic, eluding the formation of biomolecular corona. By keeping the biological identity of the virion, hybrid nanostructures maintained their natural recognition/targeting ability even in the presence of biomolecular crowding. In addition, we were able to tune the hybrid nanostructures' tropism toward E. coli based on the AuNP size. Overall, our results set the fundamental basis and a standard workflow for the development of phage-based targeting nanotools, valuable for a wide spectrum of nanotechnology applications.

M13 bacteriophages serve as a versatile foundation for nanobiotechnology due to their unique biological and chemical properties. The polypeptides that comprise their coat proteins, specifically pVIII, can be precisely tailored through genetic engineering. This enables the customized integration of various functional elements through specific interactions, leading to the development of innovative hybrid materials for applications such as energy storage, biosensing, and catalysis. Notably, a certain genetically engineered M13 bacteriophage variant, referred to as DSPH, features a pVIII with a repeating DSPHTELP peptide sequence. This sequence facilitates specific adhesion to single-walled carbon nanotubes (SWCNTs), primarily through π-π and hydrophobic interactions, though the exact mechanism remains unconfirmed. In this study, we synthesized the DSPHTELP peptide (an 8-mer peptide) and analyzed its interaction forces with different functional groups across various pH levels using surface forces apparatus (SFA). Our findings indicate that the 8-mer peptide binds most strongly to CH3 groups (Wad = 13.74 ± 1.04 mJ m-2 at pH 3.0), suggesting that hydrophobic interactions are indeed the predominant mechanism. These insights offer both quantitative and qualitative understanding of the molecular interaction mechanisms of the 8-mer peptide and clarify the basis of its specific interaction with SWCNTs through the DSPHTELP M13 bacteriophage.

A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.

Protein engineering techniques have vastly expanded their domain of impact, notably following the success of antibodies. Likewise, smaller peptide therapeutics have carved an increasingly significant niche for themselves in the pharmaceutical landscape. The concept of grafting such peptides onto larger protein scaffolds, thus harvesting the advantages of both, has given rise to a variety of protein engineering strategies that are reviewed herein. We also describe our own "Lasso-Grafting" approach, which combines traditional grafting concepts with mRNA display to streamline the production of multiple grafted drug candidates for virtually any target.

Phage display technology is a well-established versatile in vitro display technology that has been used for over 35 years to identify peptides and antibodies for use as reagents and therapeutics, as well as exploring the diversity of alternative scaffolds as another option to conventional therapeutic antibody discovery. Such successes have been responsible for spawning a range of biotechnology companies, as well as many complementary technologies devised to expedite the drug discovery process and resolve bottlenecks in the discovery workflow.

In this perspective, the authors summarize the application of phage display for drug discovery and provide examples of protein-based drugs that have either been approved or are being developed in the clinic. The amenability of phage display to generate functional protein molecules to challenging targets and recent developments of strategies and techniques designed to harness the power of sampling diverse repertoires are highlighted.

Phage display is now routinely combined with cutting-edge technologies to deep-mine antibody-based repertoires, peptide, or alternative scaffold libraries generating a wealth of data that can be leveraged, e.g. via artificial intelligence, to enable the potential for clinical success in the discovery and development of protein-based therapeutics.

Wrinkles, one of the most common signs of aging, are primarily caused by the continuous contraction of muscles. Muscle contraction is induced by the binding of acetylcholine (ACh), released at the neuromuscular junction, to nicotinic acetylcholine receptor (nAChR) present on the muscle cell surface. In this study, we aimed to develop a wrinkle-improving peptide that inhibits the binding of ACh to nAChR using peptide phage display technology. Our peptide showed a remarkably high binding affinity to nAChR subunit α1, with a value below 1 µM, and was found to inhibit the action of ACh through its interaction with these receptors. Furthermore, it increased collagen synthesis in skin cells and upregulated the expression of the aquaporin-3 (AQP3) and hyaluronan synthase-2 (HAS2) genes. These results confirm that the peptide effectively inhibits muscle contraction and enhances skin elasticity and hydration, contributing to its wrinkle-reducing effects. Clinical studies on humans observed significant improvement in wrinkles after three weeks of use, with substantial reduction observed after six weeks. In conclusion, these findings demonstrate the efficacy of the peptide (named Medipep) in reducing wrinkles.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.