Intron-mediated recombinant techniques and reagents

A combinatorial method for generating a variegated population of ribonucleic acids encoding chimeric gene products, comprising admixing a variegated set of splicing constructs under trans-splicing conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein

• EX represents an exon, said exon having a 5' exon end and a 3' exon end,
• (3'IVS) is absent or represents a 3' fragment of a group II intron, which 3' intron fragment includes a branch acceptor site and at least a portion of a domain V, and is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and
• (5'IVS) is absent or represents a 5' fragment of a group II intron, which 5' intron fragment includes an exon binding site, and is covalently attached to the 3' exon end of said exon by a phosphodiester bond,
• with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments;
• wherein said splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation and trans-splice said exons to generate a variegated library of chimeric ribonucleic acids.

A method of isolating a gene for chimeric gene product having a desired activity, comprising the steps of:

• (i) admixing a variegated population of splicing constructs under trans-splicing reaction conditions, each splicing construct comprising a ribonucleic acid represented by the general formula (3'IVS)-EX-(5'IVS), wherein
• EX represents an exon, said exon having a 5' exon end and a 3' exon end,
• (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and
• (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond,
• with the proviso that at least a portion of said splicing constructs comprise internal exons having both of said 3' and 5' intron fragments;
• wherein said trans-splicing reaction conditions comprise reaction conditions in which said 3' and 5' intron fragments reconstitute a functional intron through intermolecular complementation, which functional intron, under said trans-splicing conditions, causes ligation of said exons to generate a combinatorial library of chimeric ribonucleic acids;
• (ii) generating, from said library of chimeric ribonucleic acids, a library of replicable expression vectors;
• (iii) transforming suitable host cells with said library of replicable expression vectors;
• (iv) culturing said transformed host cells under conditions causing expression of said library of expression vectors to generate a library of gene products;
• (v) detecting, in said library of gene products, individual gene products having a desired activity;
• and
• (vi) isolating, from said library of expression vectors, genes for the gene products detected in step (v).

A splicing construct comprising a nucleic acid represented by the general Formula (3'IVS)-EX-(5'IVS), wherein

• EX represents an exon, said exon having a 5' exon end and a 3' exon end,
• (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and
• (5'IVS) represents a 5' fragment of an intron not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond,
• wherein said exon is discontinuous with any nucleic acid sequences other than said 3' and 5' intron fragments, and said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing reactions between two or more of said splicing constructs.

A library of splicing constructs comprising a variegated population of nucleic acids each represented by the general formula (3'IVS)-EX-(5'IVS), wherein

• EX represents an exon, said exon having a 5' exon end and a 3' exon end,
• (3'IVS) represents a 3' fragment of an intron not normally associated with said exon, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and
• (5'IVS) represents a 5' fragment of an intron not normally associated with said exon, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond,
• wherein said 3' and 5' intron fragments can, through intermolecular complementation, cause trans-splicing of two or more exons to each other, and said variegated population of nucleic acids is heterogeneous with respect to nucleotide sequences of said exon.

A kit for humanizing an antibody by combinatorial intron-mediated ligation, comprising a library of human framework region (FR) constructs each represented by the general formula (3'IVS)-FR-(5'IVS), wherein

• FR represents an exon encoding a human framework region selected from the group consisting of FR1, FR2, FR3 and FR4, said exon having a 5' exon end and a 3' exon end,
• (3'IVS) is absent or represents a 3' fragment of an intron, which 3' intron fragment is covalently attached to the 5' exon end of said exon by a phosphodiester bond, and
• (5'IVS) is absent or represents a 5' fragment of an intron, which 5' intron fragment is covalently attached to the 3' exon end of said exon by a phosphodiester bond,
• with the proviso that one or both of said 3' and 5' intron fragments are present in each FR construct;
• wherein said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing between said exon and a complementarity determining region (CDR) of a construct comprising CDR-encoding nucleic acid having 3' and 5' intron fragments attached thereto.