Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2000-08-25
2002-08-20
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S006120, C435S069100, C435S091100, C530S350000, C536S023100, C536S024200, C536S024300
Reexamination Certificate
active
06436665
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, the present invention relates to methods of generating fixed arrays of proteins or coded sets of protein-conjugated microparticles.
Certain macromolecules, such as proteins, are known to interact specifically with other molecules based on their three-dimensional shapes and electronic distributions. For example, proteins interact selectively with other proteins, nucleic acids, and small-molecules. The identification of molecules that interact with proteins lays the groundwork for the development of compounds to treat diseases and their associated symptoms.
The discovery of a single drug candidate can require the screening of thousands of compounds. It is therefore important to be able to screen large numbers of compounds rapidly and efficiently. One method for screening a large number of compounds is to fix candidate binding partners, such as proteins, to a solid support.
SUMMARY OF THE INVENTION
The present invention features methods for tagging or “encoding” individual in vitro translated proteins, or groups of in vitro translated proteins, with unique and minimal encoding molecules, and related methods for subsequently sorting those encoded molecules onto solid supports or microparticles. The present invention also features methods for the identification of a desired binding partner (for example, a protein or other compound) using the encoded and sorted proteins of the invention. The invention facilitates the isolation of proteins with desired properties from large pools of partially or completely random amino acid sequences. The invention also facilitates the use of automated approaches to protein or compound screening methods.
Accordingly, in a first aspect, the invention features a method for encoding and sorting an in vitro translated protein, involving the steps of providing an in vitro translated protein attached to a nucleic acid linker and attaching the protein, through the nucleic acid linker, to an encoding molecule, thereby encoding the protein.
In one embodiment, this method further involves immobilizing the encoded protein onto a solid support. In another embodiment, the candidate protein is derived from an RNA-protein fusion molecule. In yet another embodiment, the encoding molecule is made of nucleic acids, or nucleic acid analogs. Preferably, the encoding molecule comprises a unique addressing element, a linker-specific alignment element, and a linkage element between the addressing element and the linker-specific alignment element. Furthermore, the linkage element of the encoding molecule may include polyethylene glycol units (preferably, hexaethylene oxide). In yet another embodiment, the candidate protein is attached to the encoding molecule through hybridization of the linker-specific alignment element of the encoding molecule to the nucleic acid linker of the candidate protein, or to the protein itself.
In a second aspect, the invention features a method for encoding an in vitro translated protein, involving the steps of providing an in vitro translated protein and binding a nucleic acid linker to the protein, wherein the nucleic acid linker contains an addressing element, thereby encoding the protein.
In a preferred embodiment, this method further involves immobilizing he encoded protein onto a solid support. In another preferred embodiment, the candidate protein is derived from an RNA-protein fusion molecule.
In a third aspect, the invention features a method for encoding an in vitro translated protein, involving the steps of providing an in vitro translated protein and binding a nucleic acid linker to the protein, wherein an addressing element branches off from the nucleic acid linker, thereby encoding the protein.
In one embodiment, this method further involves immobilizing the encoded protein formed in the last step of the invention onto a solid support. In another embodiment, the candidate protein is derived from an RNA-protein fusion molecule. In yet another embodiment, the addressing element is bound to the nucleic acid linker by a linkage element. The linkage element of the encoding molecule may include polyethylene glycol units. Preferably, the polyethylene glycol units are hexaethylene oxide.
In yet other embodiments of each of the above aspects of the invention, the solid support is a glass or silica-based chip, or a bead. A capture probe may be attached to the solid support, and may consist of nucleic acids or nucleic acid analogs. The encoded candidate protein may be immobilized onto the solid support by hybridizing the encoded candidate protein to the nucleic acid capture probe, thus sorting the protein according to the information contained in the encoding molecule.
In further embodiments of all of the aspects of the invention, the candidate protein is labeled with a reporter tag, which is preferably a fluorophore. An affinity tag may also be attached to the encoding molecule. One exemplary affinity tag is biotin.
In yet further embodiments, the encoding molecule and solid support are functionalized with a cross-linking moiety. Preferably, the cross-linking moiety is a psoralen, azido compound, or sulfur-containing molecule. In one embodiment, he 5′ terminus of the encoding molecule is functionalized with an electrophile that cross-links regioselectively with a nucleophilic amino acid side chain of the protein.
In a fourth aspect, the invention features a method for detecting an interaction between a protein and a compound, involving the steps of providing an encoded in vitro translated protein immobilized onto a solid support; contacting the protein with a candidate compound under conditions which allow an interaction between the protein and the compound; and analyzing the solid support for the presence of the compound as an indication of an interaction between the protein and the compound. The compound may be a nucleic acid, a protein, a therapeutic, or an enzyme.
In a fifth aspect, the invention features an in vitro translated protein attached to a nucleic acid linker and bound to an encoding molecule.
In a sixth aspect, the invention features an in vitro translated protein attached to an encoded nucleic acid linker molecule.
In a seventh aspect, the invention features an in vitro translated protein attached to a branched encoded nucleic acid linker molecule.
In various preferred embodiments, the protein is attached to a solid support bearing a capture probe. In other embodiments, the encoded protein is attached to the capture probe through hybridization or a covalent bond.
As used herein, by a “protein” is meant any two or more naturally occurring or modified amino acids joined by one or more peptide bonds. “Protein,” “peptide,” and “polypeptide” are used interchangeably.
By an “encoding molecule” is meant a unique tag which may be attached to a protein or peptide and which facilitates recognition of the protein among a population of proteins. The encoding molecule may be composed of nucleic acids, nucleic acid analogs, or non-nucleosides, but it is not comprised of the RNA that, when translated, yields the protein itself. By “encode” is meant to attach an encoding molecule.
By an “addressing element” is meant that portion of an encoding molecule which gives the encoding molecule its unique identity by differing sufficiently in sequence from other such elements in a given population. Preferably the addressing element is between 4 and 40 nucleotide units in length. In addition, the addressing element may comprise nucleic acids or nucleic acid analogs.
By a “linker-specific alignment element” is meant that portion of an encoding molecule which hybridizes to the nucleic acid linker of an in vitro translated protein, or to the protein itself. The addressing element may consist of nucleic acids or nucleic acid analogs.
By a “linkage element” is meant that portion of an encoding molecule that joins the addressing element and the linker-specific alignment element together. The linkage element may be composed of nucleic acids, nucleic acid analogs, and non-nucleosides. Preferably the linkage elem
Clark & Elbing LLP
Davis Katharine F
Guzo David
Phylos, Inc
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