Hybridization control of sequence variation

A method of altering a plurality of template nucleic acid strands, the method comprising:

• a) providing i) a plurality of template nucleic acid strands and ii) a plurality of diverse nucleic acids;
• b) annealing a cleavage-directing oligonucleotide to each of the diverse nucleic acids of the plurality of diverse nucleic acids to form a cleavable region on each of the diverse nucleic acids;
• c) cleaving the cleavable region of each diverse nucleic acid to form a plurality of diversity oligonucleotides;
• d) combining the plurality of diversity oligonucleotides and the plurality of template nucleic acid strands in a mixture;
• e) subjecting the mixture to conditions such that only a subset of the plurality of diversity oligonucleotides anneal to one or more template nucleic acid strands of the plurality;
• and
• f) extending and/or ligating one or more annealed oligonucleotides of the subset to form one or more altered nucleic acid strands, each incorporating at least one of the annealed oligonucleotides and a sequence complementary to a template nucleic acid strand from the plurality.

A method of altering an immunoglobulin variable domain coding sequence, the method comprising:

• a) providing i) a template nucleic acid strand that comprises a nucleic acid sequence that encodes an amino acid sequence comprising an immunoglobulin variable domain, or a complement thereof, and ii) a plurality of diversity oligonucleotides, each diversity oligonucleotides comprising a nucleic acid sequence that encodes an amino acid sequence that includes a single complementarity determining region (CDR), and a portion of each framework region flanking the CDR, or a complement thereof;
• b) combining the plurality of diversity oligonucleotides and the template nucleic acid strand in a mixture;
• c) subjecting the mixture to conditions such that only a subset of the plurality of diversity oligonucleotides anneal to the template nucleic acid strand;
• and
• d) extending and/or ligating an annealed oligonucleotide of the subset to form an altered nucleic acid strand that is partially complementary to the template nucleic acid strand, and incorporates the annealed oligonucleotide, thereby producing an altered immunoglobulin variable domain coding sequence or complement thereof.

A method of selecting a nucleic acid encoding a protein, the method comprising:

• a) providing a plurality of genetic packages, each package comprising an accessible protein that varies among the plurality of genetic packages and a coding nucleic acid that encodes the accessible protein;
• b) selecting one or more packages of the plurality that display accessible proteins that have at least a threshold degree of a given activity;
• c) preparing template nucleic acids from at least one of the one or more selected packages;
• d) providing a plurality of diversity oligonucleotides that can anneal to at least some of the template nucleic acids;
• e) combining the diversity oligonucleotides and the template nucleic acids in a mixture;
• f) subjecting the mixture to conditions such that only a subset of the plurality of diversity oligonucleotides can anneal to the template nucleic acids;
• and
• g) extending and/or ligating an annealed oligonucleotide of the subset to form a nucleic acid strand that is partially complementary to the template nucleic acid strand and that encodes a protein having a sequence altered relative to the protein selected for at least a threshold level of a given activity.

A method of providing a library of genetic packages that present an immunoglobulin protein, the method comprising:

• a) providing a first plurality of genetic packages, each package comprising an accessible protein that comprises an immunoglobulin variable domain and varies among the plurality of genetic packages and a coding nucleic acid that encodes the accessible protein;
• b) contacting the first plurality of genetic packages to a target;
• c) separating genetic packages of the first plurality that bind to the target from genetic packages that do not bind to the target;
• d) preparing template nucleic acids from at least one of the separated genetic packages that bind to the target, the template nucleic acids comprising a sequence from the coding nucleic acid of the respective genetic packages;
• e) providing a plurality of diversity oligonucleotides that can anneal to at least some of the template nucleic acids and that each comprise a nucleic acid sequence encoding a single CDR and a portion of the flanking framework regions, or a complement thereof;
• f) combining the diversity oligonucleotides and the template nucleic acids in a mixture;
• g) subjecting the mixture to conditions such that only a subset of the plurality of diversity oligonucleotides can anneal to the template nucleic acids;
• h) extending and/or ligating an annealed oligonucleotide of the subset to form a plurality of altered nucleic acid strands that each incorporate a diversity oligonucleotide and a sequence complementary to one of the template nucleic acids, and
• i) preparing a second plurality of genetic packages from the altered nucleic acid strands or complements thereof as coding nucleic acids for the accessible protein component of each respective genetic package, thereby providing a library of genetic packages that present an immunoglobulin protein.

A method comprising:

• a) providing a plurality of diverse subject nucleic acids, each being attached to an insoluble support and including a single-stranded region;
• b) annealing a first oligonucleotide to each subject nucleic acid of the plurality to form first double-stranded segments;
• c) cleaving the first double-stranded segments to release first fragments from the insoluble support and first-cleaved subject nucleic acids attached to the insoluble support;
• d) annealing a second oligonucleotide to each of the first-cleaved subject nucleic acids to form second double-stranded segments;
• e) cleaving the second double-stranded segments to release second fragments from the insoluble support and a second-cleaved subject nucleic acid attached to the insoluble support;
• and
• f) recovering the second fragments from the insoluble support.

A method of altering a nucleic acid sequence encoding a peptide, the method comprising:

• a) providing i) one or more template nucleic acids, each encoding a peptide of less than 31 amino acids that independently binds to a target molecule, or complement thereof, and ii) a plurality of diversity oligonucleotides that can anneal to at least one of the one or more template nucleic acids at a site that overlaps a sequence encoding the peptide, or complement thereof, wherein the diversity oligonucleotides include at least 103 different nucleic acids sequences;
• b) combining the diversity oligonucleotides and the one or more template nucleic acids in a mixture;
• c) subjecting the mixture to conditions such that only a subset of the plurality of diversity oligonucleotides can anneal to the one or more template nucleic acids;
• d) extending and/or ligating an annealed oligonucleotide of the subset to form a plurality of altered nucleic acid strands that each incorporate a diversity oligonucleotide and a sequence complementary to one of the template nucleic acids.