Methods for Making and Using Molecular Switches Involving Circular Permutation
First Claim
1. A method for assembling a fusion molecule, comprising:
- generating a circular permutation of an insertion sequence; and
inserting the insertion sequence into an acceptor sequence.
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Abstract
The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.
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Citations
49 Claims
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1. A method for assembling a fusion molecule, comprising:
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generating a circular permutation of an insertion sequence; and inserting the insertion sequence into an acceptor sequence. - View Dependent Claims (2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35)
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4. A method for assembling a modulatable fusion molecule, comprising:
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generating a circular permutation of an insertion sequence; inserting the insertion sequence into an acceptor sequence, wherein the insertion sequence and the acceptor sequence each comprise a state; and selecting a fusion molecule, wherein the state of the insertion sequence and the state of the acceptor sequence are coupled. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 47)
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12. A method for assembling a multistable fusion molecule which can switch between at least an active state and a less active state, comprising:
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circularly permuting an insertion sequence; inserting the insertion sequence into an acceptor sequence, thereby generating a fusion molecule, wherein either the insertion sequence or the acceptor sequence comprises a state; and
wherein the respective other sequence is responsive to a signal; andselecting a fusion molecule, wherein the state is coupled to the signal, such that the fusion molecule switches state in response to the signal.
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30. A library, comprising a plurality of library members,
wherein each library member comprises a first nucleic acid sequence encoding a first polypeptide having a first state, the first nucleic acid sequence having been circularly permuted and inserted into a second nucleic acid sequence encoding a second polypeptide having a second state.
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36. An isolated nucleic acid encoding a molecular switch protein comprising a nucleotide sequence selected from any of SEQ ID NOS:
- 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, or an effective fragment thereof.
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37. A molecular switch protein comprising an amino acid sequence selected from any of SEQ ID NOS:
- 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, or an effective fragment thereof.
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38. A method for assembling a fusion molecule, comprising:
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generating a random circular permutation of an insertion sequence; and inserting the insertion sequence into an acceptor sequence. - View Dependent Claims (39, 40)
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41. A method for assembling a modulatable fusion molecule, comprising:
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generating a random circular permutation of an insertion sequence; inserting the insertion sequence into an acceptor sequence, wherein the insertion sequence and the acceptor sequence each comprise a state; and selecting a fusion molecule, wherein the state of the insertion sequence and the state of the acceptor sequence are coupled. - View Dependent Claims (42, 43, 44, 45, 46, 48)
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49. A method for assembling a multistable fusion molecule which can switch between at least an active state and a less active state, comprising:
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randomly circularly permuting an insertion sequence; inserting the insertion sequence into an acceptor sequence, thereby generating a fusion molecule, wherein either the insertion sequence or the acceptor sequence comprises a state; and
wherein the respective other sequence is responsive to a signal; andselecting a fusion molecule, wherein the state is coupled to the signal, such that the fusion molecule switches state in response to the signal.
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Specification