Use of a ribozyme to join nucleic acids and peptides

Details Use of a ribozyme to join nucleic acids and peptides Use of a ribozyme to join nucleic acids and peptides

2002-06-20

2004-04-06

Horlick, Kenneth R. (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S024100

Reexamination Certificate

active

06716973

ABSTRACT:

BACKGROUND OF THE INVENTION
Large combinatorial libraries of biopolymers are starting points for isolating new enzymes, binding motifs and other useful molecules. For example, current technologies can generate populations of nucleic acids with complexities on the order of 10
15
molecules and then isolate and identify a single molecule with a desired activity. Random polypeptide populations have greater chemical diversity than do polynucleotides, making them an attractive alternative to nucleic acids. Current systems are limited in their ability to easily generate large complex libraries of polypeptides that are in a form that allows the isolation and identification of rare molecules with a desired activity.
SUMMARY OF THE INVENTION
Described herein are ribozymes which join a nucleic acid (RNA or DNA) to a polypeptide, the resulting products and uses for these products. In one embodiment of the invention, a ribozyme which joins RNA to a polypeptide is used to produce a diverse library (or collection) of encoded polypeptides, in which library members are polypeptide-engineered mRNA conjugates. The polypeptide-engineered mRNA conjugates comprise an engineered mRNA, which is described below, and the translation product of the engineered mRNA. Diverse libraries of encoded polypeptides of the present invention can be screened to identify and isolate library members which are conjugates in which the polypeptide exhibits desired characteristics or properties (e.g., binding to a molecule or compound of interest, enzymatic activity). In a second embodiment, a ribozyme which joins RNA to a polypeptide is used to produce polypeptides, each of which is linked to and, thus, tagged by, a specific nucleic acid. The resulting nucleic acid-tagged polypeptides comprise a polypeptide (which is to be tagged by a nucleic acid); a peptide substrate of the ribozyme used to join the nucleic acid to the polypeptide; ribozyme RNA and a nucleic acid tag. The presence of the nucleic acid tag is useful, for example, in detecting, isolating, separating, identifying or purifying the polypeptide to which it is linked and in changing the properties (e.g., solubility) of the polypeptides. An unlimited number of nucleic acid tags can be made, which overcomes a limitation of presently-available methods, which rely on a small repertoire of affinity tags, as well as sometimes incompatible incubation protocols for each tag. Such ribozymes, methods of using them, the resulting products and methods of identifying, detecting, isolating, separating, purifying and/or using these products are described in detail herein.
One embodiment of the present invention relates to the use of ribozymes described herein to produce a diverse library or collection of encoded polypeptides in which the members are polypeptide-engineered mRNA conjugates; engineered mRNA; methods of producing diverse libraries of encoded polypeptides; methods of identifying target members of the libraries which are polypeptide-engineered mRNA conjugates which exhibit a desired activity or characteristic; target members and the components (polypeptide fragment and engineered mRNA fragment) of target members; and isolated ribozymes which join an mRNA to the translation product of the mRNA.
Polypeptide-engineered mRNA conjugates of the present invention comprise an engineered mRNA and the translation product of the engineered mRNA. Engineered mRNA of the present invention is one component of the conjugates which make up the diverse libraries of encoded polypeptides and is used to produce the conjugates. Members of the diverse library are polypeptide-engineered mRNA conjugates produced by in vitro translation of engineered mRNA. The engineered mRNA comprises: (a) a ribozyme RNA which specifically covalently links to a peptide; (b) a coding region for the peptide, referred to as a peptide tag, with which the ribozyme RNA specifically covalently links; (c) a coding region for a diverse polypeptide; and (d) two PCR primer-binding sites. In one embodiment, the engineered mRNA comprises, in order from the 5′ to the 3′ end, a ribozyme segment which comprises a first PCR primer binding-site and a motif that interacts specifically with a peptide; a coding region for the peptide with which the motif interacts; a coding region for a diverse polypeptide and a second PCR primer-binding site.
The ribozyme RNA which specifically covalently links to a peptide is present in (is a component or segment of) a ribozyme which is either a contiguous sequence (the entire ribozyme is a contiguous sequence) or comprised of two noncontiguous components: one which comprises the ribozyme RNA which specifically covalently links to the peptide and one which comprises the remainder of the ribozyme sequence. In the latter case, the ribozyme RNA which covalently links to a peptide is of sufficient length and appropriate composition to covalently link to the peptide tag in the presence of the remainder of the ribozyme sequence, under the conditions used to produce the diverse libraries. The remaining ribozyme sequence is the ribozyme sequence which, in combination with the component which covalently links to the peptide tag, makes up the complete ribozyme. The ribozyme sequence or segment which covalently links to the peptide tag can be as short as one nucleotide in length and can be from any location (e.g., 5′ end, internal segment, 3′ end) in the ribozyme. In one embodiment, the ribozyme segment includes from one to about 18 nucleotides, such as from the first to about the 18th nucleotide (from the 5′ end) of a ribozyme. In further embodiments, the ribozyme segment is the first 13 to 18 nucleotides (from the 5′ end) of the ribozyme. (e.g., the first 13, 14, 15, 16, 17, or 18 nucleotides from the 5′ end). The other component is the remaining ribozyme sequence (the remainder of the ribozyme which is necessary to form a functional ribozyme.). The two ribozyme components interact with one another to form a functional (complete) ribozyme under the conditions used to produce diverse libraries of encoded polypeptides. The 5′ end of the ribozyme RNA optionally comprises three phosphate groups or an mRNA cap, such as a 7-methyl guanosine triphosphate. Optionally, the engineered mRNA further comprises an RNA linker between the ribozyme sequence and the tag coding region. Optionally, the engineered mRNA additionally comprises a coding region for a peptide linker; this coding region is positioned between the coding region for the peptide tag and the diverse coding region. Further, the engineered mRNA can include an optional ribosome stalling site, which is located between the coding region for the diverse polypeptide segment and the second PCR primer-binding site.
In one embodiment, the engineered mRNA comprises (in order from 5′ to 3′): 1) a ribozyme sequence (also referred to as which comprises a first PCR primer-binding site and a motif that interacts specifically with a peptide; 2) a coding region for a peptide (referred to as a peptide tag), with which the ribozyme motif interacts specifically; 3) a coding region for a diverse polypeptide segment and 4) a second PCR primer-binding site. In addition, in this embodiment, the engineered mRNA can include one or more of the following optional components: 5′ phosphate groups; an RNA linker between the ribozyme sequence and the tag coding region: a coding region for a peptide linker (positioned between the tag coding region and the diverse coding region) and a ribosome stalling site which is located between the coding region for the diverse polypeptide segment and the second PCR primer-binding site.
In a specific embodiment, the motif that interacts specifically with a peptide is modified Bovine Immunodeficiency Virus-1 (BIV-1) TAR RNA and the peptide tag is one with which BIV-1 TAR RNA interacts specifically by virtue of the BIV-1 Tat sequence embedded within the tag, e.g., the Tat tag peptide 1, also referred to as the Tat tag (SEQ ID NO.: 1: MSY
SGPRPRGTRGKGRRIRR
GGK), or the Tat 2 tag

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