34results about "Directed macromolecular evolution" patented technology

Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and / or positive selection of desired properties.

In some embodiments, provided herein are compositions and methods for generating molecular expression profiles of biological samples using deterministic barcoding within tissues (DBiT-seq) for spatial omics sequencing.

The present invention provides a method for constructing a cyclic compound library that overcomes the limitations of conventional ring-closing methods for cyclic compound libraries, has the advantages of milder ring-closing reaction conditions and high generality, can be used to construct monocyclic and bicyclic compound libraries, and involves few side reactions. [Solution] A cyclic compound library and its construction method are provided, which uses a solid support, a molecule containing a photocleavable group, a linker, a building block, a ring-closing A-terminal molecule, and a ring-closing B-terminal molecule at both ends of the reaction synthesis, and utilizes the decomposition of the solid support under light irradiation to complete ring closure of the amino acid residue structures of the ring-closing A-terminal molecule A and the ring-closing B-terminal molecule B through the action of cyclohydrolase. This method, which uses mild ring closure conditions, is more universal and expands the types of chemical reactions and the diversity of the encoded compound library.

To provide a library including polynucleotide.SOLUTION: A library including polynucleotides in which at least one of polynucleotides encodes a polypeptide including a first peptide (FP), a cleavable moiety (CM), and a target binding moiety (TBM) from N-terminal to C-terminal, and the FP includes an amino acid sequence according to formula (XIII): XmCXnCXo. In the formula, m denotes 2 to 10, n denotes 3 to 10, o denotes 1 to 10, each X independently denotes amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, the CM includes at least first cleavage site, and the TBM includes an antibody chain variable region and / or an antibody heavy chain variable region.SELECTED DRAWING: None

To provide an approach for designing libraries of peptide sequences for discovery and testing of significantly more motifs than would be otherwise available in a given fixed library format.SOLUTION: An engineered peptide library comprises a plurality of peptide features, each of the peptide features including at least one peptide, the at least one peptide comprising a composite region having a defined sequence of amino acids of length N, the composite region representing k different elements, each of the different elements having a defined sequence of amino acids of length x, wherein x, N, and k are integers, wherein x is less than N, wherein k is at least 2, wherein a total number of different elements represented by the engineered peptide library is KEng, wherein the number of peptide features included in the engineered peptide library is F, and wherein KEng is greater than F.SELECTED DRAWING: Figure 1

Disclosed herein is an engineered fusion enzyme composed of a prokaryotic phage RNA polymerase (T7 RNAP) and a viral capping enzyme (NP868R) to generate RNA transcripts containing a 5' 7-methylguanylate cap.

To provide a conditionally active polypeptide having higher activity and / or selectivity in a specific environment and / or under a specific condition.SOLUTION: A non-naturally occurring polypeptide or an isolated polypeptide wherein the ratio of the activity of the assay at a first pH in presence of at least one species having a molecular weight of less than 900a. m.u. and a pKa that is a maximum of 4pH unit away from the first pH to the activity of the assay at a second pH in presence of the same at least one species is at least 1.3. The species has a pKa between the first pH and the second pH and may be a small molecule.SELECTED DRAWING: None

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro. Described are host cells comprising heterologous polynucleotides encoding polyketide synthase (PKS) variants that are capable of producing more divarinol in the presence of butyrate.

Disclosed herein is an engineered fusion enzyme composed of a prokaryotic phage RNA polymerase (T7 RNAP) and a viral capping enzyme (NP868R) for the generation of RNA transcripts containing a 5' 7-methylguanylate cap.

To provide a method for generating an antibody library.SOLUTION: (i) has at least two amino acid alterations in the light chain sequence when compared to the light chain amino acid sequence of the reference antibody, (ii) has at least two amino acid alterations in the heavy chain sequence when compared to the heavy chain amino acid sequence of the reference antibody, or (iii) has at least one amino acid alteration in the light chain sequence when compared to the light chain amino acid sequence of the reference antibody; And a variant of the reference antibody having at least one amino acid change in the heavy chain sequence when compared to the heavy chain amino acid sequence of the reference antibody, wherein each of the amino acid changes is in an amino acid residue independently encoded from a segment of the variant DNA sequence, and wherein the variant DNA segment differs from the corresponding segment of the reference antibody encoding DNA sequence by a point mutation in a DNA motif susceptible to deamination by a somatic hypermutation inducing enzyme.SELECTED DRAWING: None

A method for computational drug design using an evolutionary algorithm, comprises evaluating virtual molecules according to vector distance (VD) to at least one achievement objective that defines a desired ideal molecule. In one method the invention comprises defining a set of n achievement objectives (OA1-n), where n is at least one; defining a population (PG=0) of at least one molecule; selecting an initial population (Pparent) of at least one molecule (I1-In) from the population (PG=0); and evaluating members (I1-In) of the initial population (Pparent) against at least one of the n achievement objectives (OA1-x), where x is from 1 to n.

The present disclosure relates to methods for generating antibody libraries, antibody libraries produced using such methods, and variant antibodies. Currently, methods for improving antibody binding (affinity maturation assays) involve screening large libraries of antibody variants (often >10 nucleotides) to identify a small subset of variants with improved properties. 10 The method involves obtaining the nucleotide sequence of the framework and complementarity-determining regions of a target antibody and identifying motifs recognized by a deaminating somatic hypermutation enzyme. A small library of variants incorporating one or more of these mutations is then generated. A relatively high percentage of the variants were found to have increased affinity. The technology of the present invention has been demonstrated with trastuzumab and cathepsin S antibodies, and the variants produced are also claimed.

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9: (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

Provided herein is a method for obtaining humanized antibodies based on non-human antibodies with affinity for an antigen of interest, starting from experimental or model antibody structures, grafting non-human CDRs into a library of human frameworks, and energetically relaxing and ranking the designs using constrained / constrained atomistic simulations.

Provided is a method for screening antibodies against a specific antigen epitope, including: Incubating antigens with incorporation of photocrosslinking amino acids(designated as mutant antigen) with antibody library under light irradiation with suitable wavelength and energy, and selecting antibodies that covalently crosslink with the mutant antigen; then the antibodies selected are subjected to affinity maturation against wild-type antigen, and then epitope-directed antibodies obtained.

A method for the directed evolution of proteins. The method comprises obtaining a PCR product with a gene mutation of a target protein by means of PCR amplification; placing the PCR product in an in-vitro protein expression system for gene expression to obtain a target protein with a mutation; and performing a characterization detection on the target protein with the mutation, wherein the target protein is a target enzyme, and the characterization detection comprises detecting the activity of the target enzyme and / or the corresponding enantiomeric excess. The method not only has high stability, but also shortens the time from a generally required 2-3 weeks to about 7 hours, which greatly accelerates the evolution speed.

The present invention relates to a method for preparing a library of peptides or isolated peptides, comprising the steps of: (a) releasing one or more linear dithiol peptides, which have a sulfhydryl group at the N-terminal region of the one or more peptides and are immobilized on a solid phase via a disulfide bridge at the C-terminal region of the one or more peptides, from the solid phase with (i) an agent that reduces the disulfide bridge, thereby releasing the one or more linear dithiol peptides from the solid phase, which agent is volatile and removable by evaporation, or (ii) a base that deprotonates the sulfhydryl group at the N-terminal region of the one or more linear dithiol peptides, thereby inducing intramolecular disulfide exchange, thereby releasing the one or more linear dithiol peptides from the solid phase in the form of one or more cyclic peptides.

The present disclosure describes compositions and methods for selecting functional aptamers. In certain embodiments, provided herein are methods of using aptamer cluster-containing particles to identify functional aptamers from an aptamer library. In certain embodiments, provided herein are populations of functionally enriched aptamers. In certain embodiments, provided herein are methods of selecting aptamers for use in personalized cancer therapy and methods of creating tumor delivery systems. In certain embodiments, provided herein are compositions comprising aptamer cluster-containing particles, target cells (e.g., cancer cells, immune cells, etc.), and / or detectable indicators of cellular function (e.g., apoptosis, cell proliferation, fluorescent indicators of gene or protein expression, etc.).

The present invention provides a method for constructing a cyclic compound library that overcomes the limitations of conventional ring-closing methods for cyclic compound libraries, has the advantages of milder ring-closing reaction conditions and high generality, can be used to construct monocyclic and bicyclic compound libraries, and involves few side reactions. [Solution] A cyclic compound library and its construction method are provided, which uses a solid support, a molecule containing a photocleavable group, a linker, a building block, a ring-closing A-terminal molecule, and a ring-closing B-terminal molecule at both ends of the reaction synthesis, and utilizes the decomposition of the solid support under light irradiation to complete ring closure of the amino acid residue structures of the ring-closing A-terminal molecule A and the ring-closing B-terminal molecule B through the action of cyclohydrolase. This method, which uses mild ring closure conditions, is more universal and expands the types of chemical reactions and the diversity of the encoded compound library.

Disclosed herein is an engineered fusion enzyme composed of a prokaryotic phage RNA polymerase (T7 RNAP) and a viral capping enzyme (NP868R) for the generation of RNA transcripts containing a 5′ 7-methylguanylate cap.

According to a first aspect of the invention, a method for the generation of a cell line is provided, comprising the steps of (a) providing a plurality of mammalian B cells, wherein each of the plurality of B cells comprises a transgenic genomic DNA sequence encoding a marker protein inserted into an endogenous immunoglobulin locus comprised in said B cell, and wherein the transgenic genomic DNA sequence is amenable to cleavage by a site directed nuclease, particularly Cas9; (b) replacing the transgenic genomic DNA sequence encoding a marker protein with a second transgenic DNA sequence encoding a protein of interest; (c) sorting B cells based on the presence or absence of the marker protein; and (d) collecting B cells in which the marker protein is absent.

This disclosure relates to methods of generating antibody libraries, antibody libraries produced using such methods, and variant antibodies. Presently, methods of improving antibody binding (affinity maturation assays) require the screening of vast libraries of antibody variants (often >1010) to identify a small fraction of variants with improved characteristics. The present invention involves taking the nucleotide sequence of the framework and complementarity determining region of a target antibody and identifying motifs which would be recognised by deamination somatic hypermutation enzymes. A small library of variants is then created which incorporate one or more of these mutations. It was found that a relatively high proportion of the variants have an increased affinity. The technique of the present invention was demonstrated on the trastuzumab and Cathepsin S antibodies, and the variants produced are also claimed.

The present invention provides improved automated systems and methods for synthesis of biopolymers including DNA and RNA. The automated systems and methods represent a number of improvements over existing systems for multiplex synthesis of biopolymers in a combinatorial fashion.

This disclosure relates to methods of generating antibody libraries, antibody libraries produced using such methods, and variant antibodies. Presently, methods of improving antibody binding (affinity maturation assays) require the screening of vast libraries of antibody variants (often >1010) to identify a small fraction of variants with improved characteristics. The present invention involves taking the nucleotide sequence of the framework and complementarity determining region of a target antibody and identifying motifs which would be recognised by deamination somatic hypermutation enzymes. A small library of variants is then created which incorporate one or more of these mutations. It was found that a relatively high proportion of the variants have an increased affinity. The technique of the present invention was demonstrated on the trastuzumab and Cathepsin S antibodies, and the variants produced are also claimed.

In one aspect, the present disclosure provides a method for producing various virus-derived construct plasmids from starting plasmids (for example, the virus-derived construct plasmids of the present disclosure). In one embodiment, the method for producing virus-derived construct plasmids according to the present disclosure includes: a step for preparing a set of starting plasmids containing at least a portion of a nucleic acid sequence necessary for forming a virus-derived construct, the set of starting plasmids including a set of corresponding alternative unit nucleic acids, and the set of corresponding alternative unit nucleic acids including at least one set of corresponding alternative unit nucleic acids that contain a different nucleic acid sequence; a step for treating the set of starting plasmids with a restriction enzyme and preparing a mixed solution containing cleaved alternative unit nucleic acid fragments; a step for ligating the cleaved alternative unit nucleic acid fragments to form an integrated nucleic acid; and a step for causing the integrated nucleic acid to contact a transformed organism and thereby forming virus-derived construct plasmids.

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro. Described are host cells comprising heterologous polynucleotides encoding polyketide synthase (PKS) variants that are capable of producing more divarinol in the presence of butyrate.