Deep mutational evolution of biomolecules

A method of selecting an improved biomolecule variant, wherein the biomolecule is a protein, RNA, or DNA, comprising:

• (i) constructing a library comprising a plurality of biomolecule variants;
• wherein each variant is independently a variant of the same reference biomolecule, wherein each variant comprises an alteration of one or more monomer locations of the reference biomolecule, wherein the monomer is an amino acid of the protein or ribonucleotide of the RNA or deoxyribonucleotide of the DNA,
• wherein each alteration of a monomer location is independently selected from the group consisting of substitution of the monomer, deletion of one or more consecutive monomers beginning at the location, and insertion of one or more consecutive monomers adjacent to the location;
• and
• wherein the library represents variants comprising alteration of one or more locations of at least 5%, at least 10%, at least 30%, at least 70%, or at least 90% of the monomer locations of the reference biomolecule;
• (ii) screening the library of (i);
• (iii) identifying at least a portion of the library of (i) that exhibits one or more improved characteristics compared to the reference biomolecule;
• (iv) carrying out one or more additional rounds of library construction and screening to produce a final library, wherein construction of each library comprises:
• altering one or more additional monomer locations of the identified portion of the previous library to produce a subsequent library of biomolecule variants;
• (v) selecting the improved biomolecule variant from the final library of biomolecule variants, wherein the improved biomolecule variant exhibits one or more improved characteristics compared to the reference biomolecule.

A method of constructing a library of polynucleotide variants of a reference biomolecule, comprising:

• (a) constructing a polynucleotide that encodes for a variant of the reference biomolecule, wherein the reference biomolecule is a protein or RNA or DNA;
• wherein the polynucleotide encodes for an alteration of one or more monomer locations of the reference biomolecule, wherein the monomer is an amino acid of the protein or ribonucleotide of the RNA or the deoxyribonucleotide of the DNA, and
• wherein each alteration of a monomer location is independently selected from the group consisting of substitution of the monomer, deletion of one or more consecutive monomers beginning at the location, and insertion of one or more consecutive monomers adjacent to the location;
• and
• (b) repeating the polynucleotide construction of (a) a sufficient number of times such that the library of polynucleotide represents variants comprising a single alteration of a single location for at least of at least 5%, at least 10%, at least 30%, at least 70%, or at least 90%1% of the monomer locations of the biomolecule.

A polynucleotide variant library, comprising polynucleotide variants of a reference biomolecule, comprising:

• a plurality of polynucleotides that independently encode for a variant of the reference biomolecule, wherein the reference biomolecule is a protein or RNA or DNA;
• wherein each polynucleotide independently encodes an alteration of one or more monomer locations of the reference biomolecule, wherein the monomer is an amino acid of the protein or ribonucleotide of the RNA or deoxyribonucleotide of the DNA, and
• wherein each alteration of a monomer location is independently selected from the group consisting of substitution of the monomer, deletion of one or more consecutive monomers beginning at the location, and insertion of one or more consecutive monomers adjacent to the location;
• and
• wherein the library of polynucleotides represents variants comprising a single alteration of a single location of at least 5%, at least 10%, at least 30%, at least 70%, or at least 90% for at least 1% of the monomer locations.

A library of variant oligonucleotides, wherein:

• each variant oligonucleotide independently encodes an alteration of one or more sequential monomer locations of a reference biomolecule, wherein:
• the reference biomolecule is a protein, RNA, or DNA,
• the one or more monomers are one or more amino acids of the protein or ribonucleotides of the RNA or one or more deoxyribonucleotides of DNA, and
• wherein each alteration of a monomer location is independently selected from the group consisting of substitution of the monomer, deletion of one or more consecutive monomers beginning at the location, and insertion of one or more consecutive monomers adjacent to the location;
• each variant oligonucleotide comprises a pair of homology arms flanking the encoded alteration, wherein the homology arms are homologous to the reference biomolecule sequences flanking the corresponding monomer location alteration, and wherein each homology arm independently comprises between 10 to 100 nucleotides;
• and
• the library of variant oligonucleotides represents alteration of a single monomer for at least 80% of monomer locations.