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.

Provided herein are libraries containing synthetic polynucleotides that encode activatable binding polypeptides. Further provided herein are activatable binding polypeptides and polypeptide libraries containing such activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits, and methods of making and using activatable polypeptide libraries.

Provided herein are libraries containing synthetic polynucleotides that encode activatable binding polypeptides. Further provided herein are activatable binding polypeptides and polypeptide libraries containing such activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits, and methods of making and using activatable polypeptide libraries.

Provided herein are libraries containing synthetic polynucleotides that encode activatable binding polypeptides. Further provided herein are activatable binding polypeptides and polypeptide libraries containing such activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits, and methods of making and using activatable polypeptide libraries.

Provided herein are libraries containing synthetic polynucleotides that encode activatable binding polypeptides. Further provided herein are activatable binding polypeptides and polypeptide libraries containing such activatable binding polypeptides. Also provided herein are vectors, vector libraries, cells, kits, and methods of making and using activatable polypeptide libraries.

A general method and recombinant nucleic acid sequences, by means which the method selects a recombinant protein containing an FHA domain for binding a target molecule from a library proteins with a high-throughput method of creating protein variations within the FHA domain in non-conserved or non-structural sequences of the FHA scaffold, and the library may also be in the form of a phagemid or phage library wherein the ALP nucleic acid sequence is inserted into a vector capable of allowing the vector and expressed ALP protein from being virally packaged, and the recombinant nucleic acid sequences which are randomly mutated at varying non-conserved or non-structural FHA domain sequences.

A general method and recombinant nucleic acid sequences, by means which the method selects a recombinant protein containing an FHA domain for binding a target molecule from a library proteins with a high-throughput method of creating protein variations within the FHA domain in non-conserved or non-structural sequences of the FHA scaffold, and the library may also be in the form of a phagemid or phage library wherein the ALP nucleic acid sequence is inserted into a vector capable of allowing the vector and expressed ALP protein from being virally packaged, and the recombinant nucleic acid sequences which are randomly mutated at varying non-conserved or non-structural FHA domain sequences.

The present disclosure provides compositions, methods and systems for generating nucleic acid agents having a desired property, such as a property for specifically binding to a target. More specifically, the present disclosure provides compositions, methods and systems for generating a pool of modified members comprising modified nucleic acid agents with an unlimited range of chemical diversity.

The present disclosure relates to multifunctional molecules, including molecules according to formula (I):

([(B.sub.1).sub.M-D-L.sub.1].sub.Y-H.sub.1).sub.O-G-(H.sub.2-[L.sub.2-E-(B.sub.2).sub.K].sub.W).sub.P, (I)

wherein G, H.sub.1, H.sub.2, D, E, B.sub.1, B.sub.2, M, K, L.sub.1, L.sub.2, O, P, Y, and W are defined herein. The present disclosure also relates to methods of preparing and using such multifunctional molecules to identify encoded molecules capable of binding target molecules.

This disclosure provides methods for monitoring an immune response. Methods comprise linking a polynucleotide sequence encoding a heavy chain variable region and a polynucleotide sequence encoding a light chain variable region from a single lymphocyte from a biological sample obtained before an immune response and linking a polynucleotide sequence encoding a heavy chain variable region and a polynucleotide sequence encoding a light chain variable region from a single lymphocyte from a biological sample obtained during or after an immune response. Methods further comprise performing high-throughput sequencing of the linked (paired) sequences from before the immune response and from during or after the immune response, and comparing the resulting sequence reads.