Methods for producing 5′-nucleic acid-protein conjugates

Details Methods for producing 5′-nucleic acid-protein conjugates Methods for producing 5′-nucleic acid-protein conjugates

2000-06-01

2003-09-23

Low, Christopher S. F. (Department: 1653)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S320100, C435S091100, C435S069700, C536S023100, C424S179100, C530S395000, C530S391900, C530S391500, C514S002600

Reexamination Certificate

active

06623926

ABSTRACT:

BACKGROUND OF THE INVENTION
In general, the present invention features methods for the preparation of nucleic acid-protein conjugates.
Nucleic acid-protein conjugates, sometimes referred to as nucleic acid-protein fusions, nucleoproteins or nucleopeptides, are naturally-occurring bioconjugates which play a key role in important biological processes. In one particular example, such conjugates play a central role in the process of nucleoprotein-primed viral replication (Salas, Ann. Rev. Biochem. 60, 39-71 (1991)). Accordingly, nucleoproteins as well as nucleopeptides may serve as powerful tools for the study of biological phenomena, and may also provide a basis for the development of antiviral agents.
In addition, conjugates of peptides and nucleic acids have found use in several other applications, such as non-radioactive labels (Haralambidis et al., Nucleic Acids Res. 18, 501-505 (1990)) and PCR primers (Tong et al., J. Org. Chem. 58, 2223-2231 (1993)), as well as reagents in encoded combinatorial chemistry techniques (Nielsen et al., J.A.C.S. 115, 9812-9813 (1993)). In yet other applications, peptides predicted to have favorable interactions with cell membranes, such as polylysine (Leonetti et al., Bioconjugate Chem. 1, 149-153 (1990)), other highly basic peptides (Vives & Lebleu, Tetrahedron Lett. 328, 1183-1186 (1997)), hydrophobic peptides (Juby et al., Tetrahedron Lett. 32, 879-882 (1991)), viral fusion peptides (Soukchareun et al., Bioconjugate Chem. 6, 43-53 (1995)) and peptide signal sequences (Arar et al., Bioconjugate Chem. 6, 573-577 (1995)), have been coupled to oligonucleotides to enhance cellular uptake. Peptides able to chelate metals have also been appended to oligonucleotides to generate specific nucleic acid cleaving reagents (Truffert et al., Tetrahedron 52, 3005-3016 (1996)). And peptides linked to the 3′-end of oligonucleotides have been reported to provide important resistance to 3′-exonucleases (Juby et al., Tetrahedron Lett. 32, 879-882 (1991)).
One particular type of nucleic acid-protein conjugate, referred to as an 10. RNA-protein fusion (Szostak and, Roberts, U.S. Ser. No. 09/007,005; and Roberts'and Szostak, Proc. Natl. Acad. Sci. USA 94, 12297-12302 (1997)), has been used in methods for isolating proteins with desired properties from pools of proteins. To create such, fusions, an RNA and the peptide or protein that it encodes are joined during in vitro translation using synthetic RNA that carries a peptidyl acceptor, such as puromycin, at its 3′-end. In this process, the synthetic RNA, which is devoid of stop codons, is typically synthesized by in vitro transcription from a DNA template followed by 3′-ligation to a DNA linker carrying puromycin. The DNA template sequence causes the ribosome to pause at the 3′-end of the open reading frame, providing additional time for the puromycin to accept the nascent peptide chain and resulting in the production of the RNA-protein fusion molecule.
SUMMARY OF THE INVENTION
The present invention features chemical ligation methods for producing nucleic acid-protein conjugates in good yields. Two different approaches are described. In the first, fusions are formed by a reaction between an unprotected protein carrying an N-terminal cysteine and a nucleic acid carrying a 1,2-aminothiol reactive group. In the second approach, fusion formation occurs as the result of a bisarsenical-tetracysteine interaction.
Accordingly, in a first aspect, the invention features a method for generating a 5′-nucleic acid-protein conjugate, the method involving: (a) providing a nucleic acid which carries a reactive group at its 5′ end; (b) providing a non-derivatized protein; and (c) contacting the nucleic acid and the protein under conditions which allow the reactive group to react with the N-terminus of the protein, thereby forming a 5-nucleic acid-protein conjugate.
In a related aspect, the invention features a 5′-nucleic acid-protein conjugate which includes a nucleic acid bound through its 5′-terminus or a 5′-terminal reactive group to the N-terminus of a non-derivatized protein.
In various preferred embodiments of these aspects, the nucleic acid is greater than about 20 nucleotides in length; the nucleic acid is greater than about 120 nucleotides in length; the nucleic acid is between about 2-1000 nucleotides in length; the protein is greater than about 20 amino acids in length; the protein is greater than about 40 amino acids in length; the protein is between about 2-300 amino acids in length; the contacting step is carried out in a physiological buffer; the contacting step is carried out using a nucleic acid and a protein, both of which are present at a concentration of less than about 1 mM; the nucleic acid is DNA or RNA (for example, mRNA); the nucleic acid includes the coding sequence for the protein; the N-terminus of the non-derivatized protein is a cysteine residue; the N-terminal cysteine is exposed by protein cleavage; the reactive group is an aminothiol reactive group; the protein includes an &agr;-helical tetracysteine motif located proximal to its N-terminus; the &agr;-helical tetracysteine motif includes the sequence cys-cys-X-X-cys-cys SEQ. ID. NO: 6, wherein X is any amino acid; the reactive group is a bisarsenical derivative; the conjugate is immobilized on a solid support (for example, a bead or chip); and the conjugate is one of an array immobilized on a solid support.
In another related aspect, the invention features a method for the selection of a desired nucleic acid or a desired protein, the method involving: (a) providing a population of 5′-nucleic acid-protein conjugates, each including a nucleic acid bound through its 5′-terminus or a 5′-terminal reactive group to the N-terminus of a non-derivatized protein; (b) contacting the population of 5′-nucleic acid-protein conjugates with a binding partner specific for either the nucleic acid or the protein portion of the desired nucleic acid or desired protein under conditions which allow for the formation of a binding partner-candidate conjugate complex; and (c) substantially separating the binding partner-candidate conjugate complex from unbound members of the population, thereby selecting the desired nucleic acid or the desired protein.
In yet another related aspect, the invention features a method for detecting an interaction between a protein and a compound, the method involving: (a) providing a solid support that includes an array of immobilized 5′-nucleic acid-protein conjugates, each conjugate including a nucleic acid bound through its 5′-terminus or a 5′-terminal reactive group to the N-terminus.of a non-derivatized protein; (b) contacting the solid support with a candidate compound under conditions which allow an interaction between the protein portion of the conjugate and the compound; and (c) analyzing.the solid support for the presence of the compound as an indication of an interaction between the protein and the compound.
In various preferred embodiments of these methods, the method further involves repeating steps (b) and (c); the compound is a protein; the compound is a therapeutic; the nucleic acid is greater than about 20 nucleotides in length; the nucleic acid is greater than about 120 nucleotides in length; the nucleic acid is between about 2-1000 nucleotides in length; the protein is greater than about 20 amino acids in length; the protein is greater than about 40 amino acids in length; the protein is between about 2-300 amino acids in length; the nucleic acid is DNA or RNA (for example, mRNA); the nucleic acid includes the coding sequence for the protein, the N-terminus of the non-derivatized protein is a cysteine residue; the reactive group is an aminothiol reactive group; the protein includes an &agr;-helical tetracysteine motif located proximal to its N-terminus; the &agr;-helical tetracysteine motif includes the sequence, cys-cys-X-X-cys-cys SEQ. ID. NO: 6, wherein X is any amino acid; the reactive group is a bisarsenical derivative

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