Nucleic acid-protein fusions and methods of making and selecting fusions
View Patent ↗Described herein are RNA-protein fusion production methods which involve a high salt post-translational incubation step.
1. A molecule comprising a nucleic acid portion and a protein portion covalently bound to said nucleic acid portion through a peptide acceptor, wherein said protein portion is encoded by said nucleic acid portion and said peptide acceptor is not a tRNA and is a molecule capable of being added to the C-terminus of a growing protein chain by the catalytic activity of the ribosomal peptidyl transferase function.
2. The molecule of claim 1 , wherein said protein portion comprises two or more amino acids joined by one or more peptide bonds.
3. A method for constructing the molecule as defined in claim 1 , said method comprising (a) preparing a DNA containing a protein coding sequence; (b) transcribing the DNA into RNA; (c) covalently bonding to the 3′ end of the protein coding sequence a peptide acceptor; and (d) translating the RNA in a cell-free protein synthesis system, thereby constructing the molecule of claim 1 .
4. The method of claim 3 , wherein step (a) comprises synthesizing a DNA primer and a DNA template, and amplifying said DNA template using said DNA primer via polymerase chain reaction.
5. The method of claim 3 , wherein said cell-free protein synthesis system in step (d) is a wheat germ system or a reticulocyte system.
6. A method for in vitro selection and evolution, wherein said method comprises the steps of:
(a) constructing a first plurality of molecules, wherein each molecule is a molecule according to claim 1 ;
(b) selecting one or more molecules from said plurality, thereby obtaining one or more first selected molecules;
(c) using the nucleic acid portion of the one or more first selected molecules to mutagenically construct a second plurality of molecules, wherein each molecule is a molecule according to claim 1 .
7. The method according to claim 6 , further comprising the additional step of selecting one or more molecules from said second plurality, thereby obtaining one or more second selected molecules, wherein the nucleic acid and protein portions of said one or more second selected molecules differ from the nucleic acid and protein portions of said one or more first selected molecule.
8. The method according to claim 6 , wherein said selecting steps comprises contacting said first plurality of molecules with a target molecule or immobilized selection motif.
9. The method according to claim 7 , wherein said selecting steps are carried out by contacting said first and second plurality of molecules with a target molecule or immobilized selection motif.
10. The method according to any of claims 6 - 9 , wherein step (c) comprises amplification via mutagenic PCR.
11. A method for assaying protein/protein or protein/nucleic acid interaction, which comprises the steps of (a) constructing a molecule according to claim 1 , and (b) determining whether said molecule interacts with another protein or nucleic acid, thereby assaying protein/protein or protein/nucleic acid interaction.
12. The method of claim 11 , wherein step (b ) is carried out by combining the molecule according to claim 1 with an antibody, and determining whether said antibody binds to the protein portion of said molecule.
13. The method according to any of claims 11 and 12 , wherein said step (b) comprises an immunoprecipitation reaction.
14. The method according to claim 13 , wherein said immunoprecipitation reaction is carried out with a c-myc antibody.
15. The molecule according to claim 1 , wherein the peptide acceptor is puromycin.