Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-07-19
2002-08-06
Yucel, Remy (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S068100, C435S091500
Reexamination Certificate
active
06429300
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, the present invention relates to ligation methods, in particular, for joining peptide acceptors to nucleic acids. Methods currently exist for the preparation of RNA-protein fusions. An RNA-protein fusion is created by attaching a peptide acceptor to the 3′ end of an RNA molecule, followed by in vitro or in situ translation of the RNA. The product is a peptide attached to the 3′ end of the RNA encoding it. The generation of these RNA-protein fusions facilitates the isolation of proteins with desired properties from large pools of partially or completely random amino acid sequences, and solves the problem of recovering and amplifying protein sequence information by covalently attaching the RNA coding sequence to its corresponding protein molecule.
SUMMARY OF THE INVENTION
The present invention features methods for the attachment of a peptide acceptor to an RNA molecule as well as the RNA-peptide acceptor products. These methods facilitate the production of RNA-protein fusions which can be used, for example, for the isolation of proteins or nucleic acids with desired properties from large pools of partially or completely random amino acid or nucleic acid sequences. This inventive method may be carried out by a variety of strategies for affixing a peptide acceptor to a nucleic acid molecule. These various approaches differ from one another in the types of bonds formed by the attachment of the peptide to the nucleic acid, and in the reagents used to achieve the attachment.
Accordingly, in a first aspect, the invention features a method for affixing a peptide acceptor to an RNA molecule involving providing an RNA molecule having a 3′ sequence which forms a hairpin structure, providing a peptide acceptor covalently bonded to a nucleic acid linker molecule, and hybridizing the RNA molecule to the nucleic acid linker molecule under conditions which allow covalent bond formation to occur between the peptide acceptor and the RNA molecule.
In a second aspect, the invention features a method for affixing a peptide acceptor to an RNA molecule involving providing a peptide acceptor having a linker with a 5′ sequence that forms a hairpin, hybridizing the peptide acceptor to the RNA molecule, and covalently bonding the peptide acceptor to the RNA. In one embodiment of the above aspects of the invention, the peptide acceptor is bonded to the RNA molecule using T4 DNA ligase.
In a third aspect, the invention features a method for attaching a peptide acceptor to an RNA molecule, by providing an RNA molecule and a peptide acceptor covalently bonded to a linker molecule, where the linker molecule initiates with a deoxynucleotide triphosphate or dideoxynucleotide triphosphate, and contacting the RNA molecule and peptide acceptor with terminal deoxynucleotidyl transferase to covalently bond the peptide acceptor to the RNA molecule.
In a fourth aspect, the invention features a method of affixing a peptide acceptor to an RNA molecule by chemically ligating the RNA molecule to the peptide acceptor.
In one embodiment of this aspect, the peptide acceptor is joined to a psoralen moiety and crosslinked to the RNA molecule via the psoralen moiety. The psoralen moiety may be attached to either the 5′ or 3′ end of a linker molecule which is itself attached to the peptide acceptor, or the psoralen moiety may be located at an internal position of the linker molecule. According to this technique, the peptide acceptor is crosslinked to the RNA molecule using UV irradiation. In further embodiments of this particular aspect, the psoralen is attached to the peptide acceptor through a C6 alkyl chain and/or the RNA molecule contains a stop codon positioned proximal to its 3′ end. Preferably, the linker is between 25 and 40 nucleotide units in length. In addition, prior to crosslinking the peptide acceptor to the RNA molecule, the RNA may be hybridized to a linker that further includes a photocleavable moiety. The hybridized RNA may then be immobilized to a solid support through the photocleavable moiety. Preferably, the photocleavable moiety is biotin.
In another embodiment of the fourth aspect of the invention, the RNA molecule is functionalized and is attached to a peptide that has been suitably modified to permit chemical bond formation between the peptide acceptor and the RNA molecule. Preferably, the RNA molecule is functionalized through IO
4
−
oxidation. The peptide acceptor may be functionalized by attaching a molecule to the peptide acceptor chosen from the group consisting of amines, hydrazines, (thio)hydrazides, and (thio)semicarbazones.
In yet another embodiment of the fourth aspect of the invention, the chemical ligation is carried out in the absence of an external template. Alternatively, the chemical ligation reaction can be carried out in the presence of an external template. This second method involves aligning the RNA molecule and the linker portion of a peptide acceptor using a template, so that the 5′ end of the template hybridizes to the linker portion of the peptide acceptor and the 3′ end of the template hybridizes to the RNA molecule. The chemical ligation of an RNA molecule to a peptide acceptor can also be carried out in the absence of an external template by hybridizing the linker molecule itself, which is covalently bonded to the peptide acceptor, to the RNA molecule. This hybridization brings the peptide acceptor and RNA molecule into close proximity for ligation. Preferably, the functional group is at the 5′ end of the linker region of the peptide acceptor, or is flanked by a hybridization domain on one side and the peptide acceptor on the other side.
In a further embodiment of the fourth aspect of the invention, the chemical ligation of the peptide acceptor to the RNA molecule involves attaching a functional group to the RNA molecule through reductive amination of the RNA, followed by modification of the peptide acceptor to react with the RNA molecule. The two molecules are then joined through formation of a covalent bond. Preferably, the functional group attached to the RNA molecule is a thiol, maleimide, or amine.
In a fifth aspect, the invention features a method for attaching a peptide acceptor to an RNA molecule through a non-covalent bond. In one embodiment, the attachment is achieved by covalently bonding a peptide nucleic acid (PNA) to the peptide acceptor and non-covalently bonding the peptide acceptor to the RNA molecule through the PNA. In this embodiment, the RNA molecule may contain a stop codon.
In yet other aspects, the invention features RNA molecules chemically or non-covalently ligated to peptide acceptors as well as the nucleic acid-protein fusions generated by transcription and translation (and, if desired, reverse transcription and/or amplification) of these RNA molecules. In one embodiment, the peptide acceptor is ligated at the 3′ end of the RNA molecule.
In still another aspect, the invention features methods for the selection of a desired protein or nucleic acid using the RNA-peptide acceptor molecules of the invention. The selection techniques utilize the present molecules for RNA-protein fusion formation, and subsequent selection of proteins or nucleic acids of interest. The selection methods may be carried out by any of the approaches described, for example, in Szostak et al., WO 98/31700, and Szostak et al., U.S. Ser. No. 09/247,190 now U.S. Pat. No. 6,261,804, hereby incorporated by reference.
In a final aspect, the invention features a method of generating an RNA-protein fusion. This method involves providing an RNA molecule hybridized to a linker, where the linker contains a photocleavable moiety, a psoralen moiety, and a peptide acceptor; immobilizing the RNA to a solid support under conditions in which non-immobilized RNA are substantially removed from the support; crosslinking the peptide acceptor to the RNA, through the psoralen moiety, whereby this crosslinking simultaneously releases the crosslinked RNA from the solid support; and translati
Kurz Markus
Lohse Peter
Wagner Richard
Clark & Elbing LLP
Loeb Bronwen M.
Phylos, Inc.
Yucel Remy
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