Generation Of Libraries Research Articles

Transformer-Decoder GPT Models for Generating Virtual Screening Libraries of HMG-Coenzyme A Reductase Inhibitors: Effects of Temperature, Prompt Length, and Transfer-Learning Strategies.

Attention-based decoder models were used to generate libraries of novel inhibitors for the HMG-Coenzyme A reductase (HMGCR) enzyme. These deep neural network models were pretrained on previously synthesized drug-like molecules from the ZINC15 database to learn the syntax of SMILES strings and then fine-tuned with a set of ∼1000 molecules that inhibit HMGCR. The number of layers used for pretraining and fine-tuning was varied to find the optimal balance for robust library generation. Virtual screening libraries were also generated with different temperatures and numbers of input tokens (prompt length) to find the most desirable molecular properties. The resulting libraries were screened against several criteria, including IC50 values predicted by a dense neural network (DNN) trained on experimental HMGCR IC50 values, docking scores from AutoDock Vina (via Dockstring), a calculated quantitative estimate of druglikeness, and Tanimoto similarity to known HMGCR inhibitors. It was found that 50/50 or 25/75% pretrained/fine-tuned models with a nonzero temperature and shorter prompt lengths produced the most robust libraries, and the DNN-predicted IC50 values had good correlation with docking scores and statin similarity. 42% of generated molecules were classified as statin-like by k-means clustering, with the rosuvastatin-like group having the lowest IC50 values and lowest docking scores.

High‐Throughput Absorbance‐Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases

AbstractRecent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy‐intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet‐based microfluidic systems have emerged as a powerful high‐throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet‐based microfluidic systems, we present an absorbance‐activated droplet sorter that allows droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105‐member aldehyde dehydrogenase library towards D‐glyceraldehyde using a NADH mediated coupled assay that generates WST‐1 formazan as the colorimetric product. We successfully identify a variant with a 51 % improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Spreading Sankofa

Epistemicide is the devaluing, silencing, or annihilation of knowledge and encompasses systematic knowledge destruction enacted through accumulation of epistemic injustices. Considering prior epistemicide research in LIS––in conjunction with critical, historical, and philosophical perspectives of technology––this paper examines how epistemicide occurs by and through digital technologies, constituting what we call digital epistemicide. The social, cultural, and historical epistemic injustices enabled through technology––and the proliferation of epistemic injustices in digital worlds––demands a theorization of how epistemicide occurs in digital contexts. By considering elements of power, domination, and control in relation to digital technologies, we contribute to a critically informed meta-language for describing digital epistemicide. With concern and care for the next generation of library and information professionals, we call for an enactment of Sankofa Interventions. These include everyday dialogues, practices, policies, actions, or critical reflections that engage with historical narratives from the past, absent from discourses of the present, in a manner that emulates the notion of Sankofa. The enactment of Sankofa interventions to mitigate digital epistemicide, such as reparative storytelling and other critical digital pedagogical practices, requires both ongoing attention to digitizing records that have been left behind and likewise ensuring that digital worlds are well maintained to support the preservation and sharing of knowledge.

Multi-Mode Data Organization and File Retrieval Based on a Primer Library in Large-Scale Digital DNA Storage

At present, the polymerase chain reaction (PCR) amplification-based file retrieval method is the most commonly used and effective means of DNA file retrieval. The number of orthogonal primers limits the number of files that can be accurately accessed, which in turn affects the density in a single oligo pool of digital DNA storage. In this paper, a multi-mode DNA sequence design method based on PCR file retrieval in a single oligonucleotide pool is proposed for high-capacity DNA data storage. Firstly, by analyzing the maximum number of orthogonal primers at each predicted primer length, it was found that the relationship between primer length and the maximum available primer number does not increase linearly, and the maximum number of orthogonal primers is on the order of 104. Next, this paper analyzes the maximum address space capacity of DNA sequences with different types of primer binding sites for file mapping. In the case where the capacity of the primer library is R (where R is even), the number of address spaces that can be mapped by the single-primer DNA sequence design scheme proposed in this paper is four times that of the previous one, and the two-level primer DNA sequence design scheme can reach [R2∙(R2-1)]2 times. Finally, a multi-mode DNA sequence generation method is designed based on the number of files to be stored in the oligonucleotide pool, in order to meet the requirements of the random retrieval of target files in an oligonucleotide pool with large-scale file numbers. The performance of the primers generated by the orthogonal primer library generator proposed in this paper is verified, and the average Gibbs free energy of the most stable heterodimer formed between the orthogonal primers produced is –1 kcal∙(mol∙L−1)−1 (1 kcal = 4.184 kJ). At the same time, by selectively PCR-amplifying the DNA sequences of the two-level primer binding sites for random access, the target sequence can be accurately read with a minimum of 103 reads, when the primer binding site sequences at different positions are mutually different. This paper provides a pipeline for orthogonal primer library generation and multi-mode mapping schemes between files and primers, which can help achieve precise random access to files in large-scale DNA oligo pools.

Molli: A General Purpose Python Toolkit for Combinatorial Small Molecule Library Generation, Manipulation, and Feature Extraction.

The construction, management, and analysis of large in silico molecular libraries is critical in many areas of modern chemistry. Herein, we introduce the MOLecular LIibrary toolkit, "molli", which is a Python 3 cheminformatics module that provides a streamlined interface for manipulating large in silico libraries. Three-dimensional, combinatorial molecule libraries can be expanded directly from two-dimensional chemical structure fragments stored in CDXML files with high stereochemical fidelity. Geometry optimization, property calculation, and conformer generation are executed by interfacing with widely used computational chemistry programs such as OpenBabel, RDKit, ORCA, NWChem, and xTB/CREST. Conformer-dependent grid-based feature calculators provide numerical representation and interface to robust three-dimensional visualization tools that provide comprehensive images to enhance human understanding of libraries with thousands of members. The package includes a command-line interface in addition to Python classes to streamline frequently used workflows. Parallel performance is benchmarked on various hardware platforms, and common workflows are demonstrated for different tasks ranging from optimized grid-based descriptor calculation on catalyst libraries to an NMR chemical shift prediction workflow from CDXML files.

Carafe enables high quality in silico spectral library generation for data-independent acquisition proteomics.

Data-independent acquisition (DIA)-based mass spectrometry is becoming an increasingly popular mass spectrometry acquisition strategy for carrying out quantitative proteomics experiments. Most of the popular DIA search engines make use of in silico generated spectral libraries. However, the generation of high-quality spectral libraries for DIA data analysis remains a challenge, particularly because most such libraries are generated directly from data-dependent acquisition (DDA) data or are from in silico prediction using models trained on DDA data. In this study, we developed Carafe, a tool that generates high-quality experiment-specific in silico spectral libraries by training deep learning models directly on DIA data. We demonstrate the performance of Carafe on a wide range of DIA datasets, where we observe improved fragment ion intensity prediction and peptide detection relative to existing pretrained DDA models.

Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (VH) scaffold and four distinct variable light (VL) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a VL-linker-VH orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.

Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.

Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Generation of Antibody Libraries for Phage Display: Human Fab Format.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains VH and VL (Vκ or Vλ), fused to the human constant domains CH1 of IgG1 and CL (Cκ or Cλ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (VH, Vκ, and Vλ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.

Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent E. coli.

The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming Escherichia coli with plasmids, including phagemids. Here, we describe the preparation of electrocompetent E. coli for the generation of phagemid-encoded antibody libraries encompassing 109-1011 independent transformants. To become electrocompetent, the bacterial suspension has to have high resistance, i.e., low ionic strength, which is achieved by gradually and gently transferring bacteria grown to mid-log phase to 10% (v/v) glycerol in highly pure water. The electrocompetent E. coli must be F plasmid-harboring bacteria, referred to as F+ or male, in order to express F pili and be susceptible to infection by filamentous phage during library generation. In addition, it is necessary to apply antibiotic (e.g., tetracycline) pressure to retain the F plasmid, as it tends to segregate from bacteria. This protocol also includes assays for analyzing the prepared electrocompetent E. coli for competency, and evaluating potential contamination with helper phage, phagemid and phagemid-derived filamentous phage, and lytic phage.

Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (VH, Vκ, and Vλ) fused to human constant domains. The human constant domains, CH1 of IgG1 and CL (Cκ or Cλ), not only provide established purification and detection handles but also facilitate higher expression in Escherichia coli compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.

Generation of Antibody Libraries for Phage Display: Library Reamplification.

Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible. This is because reamplification will bias the Fab library for clones that are expressed at higher rates. Fab-phage, however, should only be used if they have been prepared on the same day, to avoid proteolytic cleavage of the physical linkage of phenotype (phage-displayed Fab protein) and genotype (phage-encapsulated Fab DNA). Thus, in practice, reamplification of a Fab-phage library cannot usually be avoided. Here, we describe the steps for the reamplification of an original Fab-phage library prior to its selection. The protocol can also be used to reamplify Fab-phage from the third or later panning rounds when enriched clones are unlikely to be lost by reamplification biases.

Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.

The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.

IDEIS: a tool to identify PTPRC/CD45 isoforms from single-cell transcriptomic data.

Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.

FastAd: A versatile toolkit for rapid generation of single adenoviruses or diverse adenoviral vector libraries

Adenoviruses (Ads) are potent gene delivery vectors for in vitro and in vivo applications. However, current methods for their construction are time-consuming and inefficient, limiting their rapid production and utility in generating complex genetic libraries. Here, we introduce FastAd, a rapid and easy-to-use technology for inserting recombinant “donor” DNA directly into infectious “receiver” Ads in mammalian cells by the concerted action of two efficient recombinases: Cre and Bxb1. Subsequently, the resulting mixed recombinant Ad population is subjected to negative selections by flippase (FLP) recombinase to remove viruses that missed the initial recombination. With this approach, recombinant Ad production time is reduced from two months to 10 days or less. FastAd can be applied for inserting complex genetic DNA libraries into Ad genomes, as evidenced by the generation of barcode libraries with over 3 million unique clones from a T25 flask-scale of transfection. Furthermore, we leveraged FastAd to construct an Ad library containing a comprehensive genome-wide CRISPR/Cas9 guide RNA (gRNA) library and demonstrated its effectiveness in uncovering novel virus-host interactions. In summary, FastAd enables the rapid generation of single Ad vectors or complex genetic libraries, facilitating not only novel applications of Ad vectors but also research in foundational Ad biology.

Computational Analysis of T-Cell Receptor Repertoire Workflow: From T-Cell Isolation to Bioinformatics Analysis.

The T-cell receptor (TCR) is the key molecule involved in the adaptive immune response. It is generated by the V(D)J recombination, responsible of the enormous diversity of the TCR repertoire, a crucial feature determining the individual capability to response to antigens and to build immunological memory. A pivotal role in the recognition of antigen is played by the hypervariable complementarity-determining region 3 (CDR3) of the V-beta chain of TCR. Investigating the CDR3 supports the understanding of the adaptive immune system dynamics in physiological processes, such as immune aging, and in disease, especially autoimmune disorders in which T cells are main actors. High-throughput sequencing (HTS) paved the way for a great progress in the investigation of TCR repertoire, enhancing the read depth in the process of library generation of sequencing and the number of samples that can be analyzed simultaneously. Therefore, the leverage of big datasets stressed the need to develop computational approach, by bioinformatics, to unravel the characteristics of the TCR repertoire.

Barcoding of Italian mosquitoes (BITMO): generation and validation of DNA barcoding reference libraries for native and alien species of Culicidae

BackgroundMosquitoes (Culicidae), as disease vectors, represent a risk for human health worldwide. Repeated introductions of alien mosquito species and the spread of invasive species have been recorded in different countries. Traditionally, identification of mosquitoes relies on morphological observation. However, morphology-based identification is associated with a number of potential disadvantages, such as the high level of specialisation of the operator and its limited applicability to damaged samples. In these cases, species identification is achieved through molecular methods based on DNA amplification. Molecular-based taxonomy has also enabled the development of techniques for the study of environmental DNA (eDNA). Previous studies indicated the 16S mitochondrial ribosomal RNA (rRNA) gene as a promising target for this application; however, 16S rRNA sequences are available for only a limited number of mosquito species. In addition, although primers for the 16S rRNA gene were designed years ago, they are based on limited numbers of mosquito sequences. Thus, the aims of this study were to: (i) design pan-mosquito 16S rRNA gene primers; (ii) using these primers, generate a 16S rRNA gene mosquito reference library (with a focus on mosquitoes present in Italy); and (iii) compare the discriminatory power of the 16S rRNA gene with two widely used molecular markers, cytochrome c oxidase subunit 1 mitochondrial gene (COI) and internal transcribed spacer 2 (ITS2).MethodsA total of six mosquito genera (28 mosquito species) were included in this study: Aedes (n = 16 species), Anopheles (5 species), Coquillettidia (1 species), Culex (3 species), Culiseta (2 species) and Uranotaenia (1 species). DNA was extracted from the whole mosquito body, and more than one specimen for each species was included in the analysis. Sanger sequencing was used to generate DNA sequences that were then analysed through the Barcode of Life Data Systems (BOLD). Phylogenetic analyses were also performed.ResultsNovel 16S rDNA gene, COI and ITS2 sequences were generated. The 16S rRNA gene was shown to possess sufficient informativeness for the identification of mosquito species, with a discriminatory power equivalent to that of COI.ConclusionsThis study contributes to the generation of DNA barcode libraries, focussed on Italian mosquitoes, with a significant increase in the number of 16S rRNA gene sequences. We hope that these novel sequences will provide a resource for studies on the biodiversity, monitoring and metabarcoding of mosquitoes, including eDNA-based approaches.Graphical abstract

Introducing synthetic thermostable RNase inhibitors to single-cell RNA-seq

Single-cell RNA-sequencing (scRNAseq) is revolutionizing biomedicine, propelled by advances in methodology, ease of use, and cost reduction of library preparation. Over the past decade, there have been remarkable technical improvements in most aspects of single-cell transcriptomics. Yet, little to no progress has been made in advancing RNase inhibition despite maintained RNA integrity being critical during cell collection, storage, and cDNA library generation. Here, we demonstrate that a synthetic thermostable RNase inhibitor (SEQURNA) yields single-cell libraries of equal or superior quality compared to ubiquitously used protein-based recombinant RNase inhibitors (RRIs). Importantly, the synthetic RNase inhibitor provides additional unique improvements in reproducibility and throughput, enables new experimental workflows including retained RNase inhibition throughout heat cycles, and can reduce the need for dry-ice transports. In summary, replacing RRIs represents a substantial advancement in the field of single-cell transcriptomics.