METHODS FOR SCREENING PROTEINS USING DNA ENCODED CHEMICAL LIBRARIES AS TEMPLATES FOR ENZYME CATALYSIS
First Claim
1. A method for screening a protein library for a protein having a desired activity, the method comprising:
- a) providing a plurality of nucleic acids, each nucleic acid having a predefined sequence, at a distinct feature of a solid support;
b) expressing in vitro a plurality of proteins from the plurality of nucleic acids;
c) exposing each of the plurality of proteins with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore;
d) detecting the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes; and
e) isolating the complexes showing detectable fluorescence energy transfer, thereby isolating the proteins having the desired enzymatic activity.
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Abstract
Disclosed are methods, compositions and devices for screening a protein library for proteins having a desired activity, such as capable of catalyzing the formation of a bond between two reactants. In an exemplary embodiments, a plurality of proteins are expressed in vitro from a plurality of nucleic acids, the plurality of proteins are exposed with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore, the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes is detected and the complexes showing detectable fluorescence energy transfer are isolated, thereby isolating proteins having the desired enzymatic activity.
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Citations
18 Claims
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1. A method for screening a protein library for a protein having a desired activity, the method comprising:
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a) providing a plurality of nucleic acids, each nucleic acid having a predefined sequence, at a distinct feature of a solid support; b) expressing in vitro a plurality of proteins from the plurality of nucleic acids; c) exposing each of the plurality of proteins with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore; d) detecting the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes; and e) isolating the complexes showing detectable fluorescence energy transfer, thereby isolating the proteins having the desired enzymatic activity. - View Dependent Claims (2, 3, 4, 5)
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6. A method for screening a protein library for a protein having desired activity, the method comprising:
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a) providing a plurality of nucleic acids, each nucleic acid having a predefined sequence, at a distinct feature of a solid support; b) expressing in vitro a plurality of proteins from the plurality of nucleic acids; c) exposing each of the plurality of proteins with two oligonucleotides, each oligonucleotide having a reactant at one end, wherein proteins having the desired activity bind the two reactants thereby forming a complex with the two oligonucleotides; and d) providing a ligase, wherein the ligase catalyses the ligation of free ends of the two oligonucleotides complexed with the proteins having the desired activity. - View Dependent Claims (7, 8, 9, 10)
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11. A method for screening a protein library for a protein having a desired activity, the method comprising:
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a) incubating a host cell comprising a first nucleic acid sequence encoding a plurality of proteins fused to aga-2 and a second nucleic acid sequence encoding a plurality of transcription activator like-effector fused with aga-1, under conditions sufficient for expressing the protein and the transcription activator like-effector as a protein complex at the cell surface; b) exposing the protein complex to a first single stranded oligonucleotide having a first reactant, wherein the oligonucleotide binds to the transcription activator like-effector; c) exposing the protein complex bound to the transcription activator like-effector, to a second single stranded oligonucleotide having a second reactant and a detectable label, wherein the second single stranded oligonucleotide has a sequence complementary to the first single stranded oligonucleotide; and d) detecting the label at the surface of the host cell, wherein the detection of the label is correlated to a presence of the protein having the desired activity. - View Dependent Claims (12, 13)
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14. A method for screening a protein library for a protein having a desired activity, the method comprising:
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a) providing a solid surface having a plurality of anchor oligonucleotides capable of hybridizing with a plurality of double stranded nucleic acids encoding a plurality of proteins; b) hybridizing a single stranded oligonucleotide having a first reactant to a free end of the double stranded nucleic acids; c) expressing in vitro a plurality of proteins from the plurality of double stranded nucleic acids in presence of a reactant having a detectable label; and d) identifying features having the detectable label, wherein the detection of the label is correlated to a presence of the proteins having the desired activity. - View Dependent Claims (15, 16, 17, 18)
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Specification