Intron-mediated recombinant techniques and reagents

A method for generating a chimeric ribonucleic acid by trans-splicing, comprising admixing two or more splicing constructs under trans-splicing reaction conditions, which splicing constructs comprise a ribonucleic acid represented by the general formula (3' IVS)-EX-(5' IVS), wherein

• EX represents an exonic ribonucleic acid sequence which is intended to be present in a chimeric ribonucleic acid, said exonic sequence 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 exonic sequence 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 exonic sequence by a phosphodiester bond,
• with the proviso that
• at least one of (3' IVS) and (5' IVS) is present on each splicing construct;
• and
• the intron fragments for at least a portion of said splicing constructs are not from intronic sequences which naturally flank the corresponding covalently attached exonic sequence;
• wherein said exons of said set of splicing constructs comprise a variegated population of ribonucleic acids, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate said chimeric ribonucleic acid.

A method for generating a chimeric ribonucleic acid by trans-splicing, comprising admixing a two or more splicing constructs under trans-splicing reaction conditions, which splicing constructs comprise a ribonucleic acid represented by the general formula (3' IVS)-EX-(5' IVS), wherein

• EX represents an ribonucleic acid encoding at least a portion of a mammalian polypetide, 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 one of (3' IVS) and (5' IVS) is present on each splicing construct,
• wherein said exons of said set of splicing constructs comprise a variegated population of polypeptide-encoding sequences, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate said chimeric ribonucleic acid.

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 exonic ribonucleic acid which is intended to be present in a chimeric ribonucleic acid, 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 one of (3' IVS) and (5' IVS) is present on each splicing construct;
• and
• the intron fragments for at least a portion of said splicing constructs are not from intronic sequences which naturally flank the corresponding covalently attached exon;
• wherein said exons of said set of splicing constructs comprise a variegated population of ribonucleic acids, and said trans-splicing reaction conditions comprise conditions in which 3' and 5' intron fragments of different splicing constructs reconstitute a functional intron through intermolecular complementation and ligate said exons to generate said chimeric ribonucleic acid.

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

• EX represents an ribonucleic acid encoding at least a portion of a mammalian polypetide, 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 one of (3' IVS) and (5' IVS) is present on each splicing construct,
• wherein said exon sequence is discontinuous with any nucleic acid sequences other than said flanking intron sequences, and said 3' and 5' intron fragments can, through intermolecular complementation, mediate trans-splicing reactions between two or more of said splicing constructs.