Methods and compositions for use in spliceosome mediated RNA trans-splicing

A cell comprising a nucleic acid molecule wherein said nucleic acid molecule comprises:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 5' splice site;
• c) a spacer region that separates the 5' splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding, domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site;
• c) a spacer region that separates the 3' splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A cell comprising a recombinant vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 5' splice site;
• c) a spacer region that separates the 5' splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A method of producing a chimeric RNA molecule in a cell comprising:

• contacting a target pre-mRNA expressed in the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site;
• c) a spacer region that separates the 3' splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric RNA within the cell.

A method of producing a chimeric RNA molecule in a cell comprising:

• contacting a target pre-mRNA expressed within the cell with a nucleic acid molecule recognized by nuclear splicing components wherein said nucleic acid molecule comprises:
• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 5' splice site;
• c) a spacer region that separates the 5' splice site from the target bindings domain;
• and
• (d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 3' splice region comprising a branch point a pyrimidine tract and a 3' splice acceptor site;
• c) a spacer region that separates the 3' splice region from the target binding domain;
• d) a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 3' splice site;
• and
• e) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 5' splice site;
• c) a spacer region that separates the 5' splice site from the target blinding domain;
• d) a safety sequence comprising one or more complementary sequences that bind to one or both sides of the 5' splice site;
• and
• e) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site;
• c) a spacer region that separates the 3' splice region from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A eukaryotic expression vector wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 5' splice site;
• c) a spacer region that separates the 5' splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

An expression library comprising recombinant expression vectors wherein said vectors expresses a nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 3' splice region comprising a branch point, a pyrimidine tract and a 3' splice acceptor site;
• c) a spacer region that separates the 3' splice region from the target binding domain;
• and
• (d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

An expression library comprising recombinant expression vectors wherein said wherein said vector expresses a nucleic acid molecule comprising:

• a) one or more target binding domains wherein the target binding domain is a random nucleotide sequence that targets binding to a pre-mRNA expressed within the cell;
• b) a 5' splice site;
• c) a spacer region that separates the 5' splice site from the target binding domain;
• and
• d) a nucleotide sequence to be trans-spliced to the target pre-mRNA;
• wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell.

A method for mapping exon-intron boundaries in pre-mRNA molecules comprising:

• (i) contacting a nucleic acid molecule containing a target binding domain wherein the target binding domain is a random nucleotide sequence that targets binding to a target pre-mRNA molecule, under conditions in which a portion of the nucleic acid molecule is trans-spliced to a portion of the target pre-mRNA to form a chimeric mRNA;
• (ii) amplifying the chimeric mRNA molecule;
• (iii) selectively purifying the amplified molecule;
• and
• iv) determining the nucleotide sequence of the amplified molecule thereby identifying the intron-exon boundaries.

A method for identifying proteins expressed in a cell comprising:

• (i) contacting a nucleic acid molecule containing a nucleotide sequence encoding a peptide tag or marker and a target binding domain wherein the target binding domain is a random nucleotide sequence with a pre-mRNA molecule under conditions in which a portion of the pre-trans-splicing molecule is trans-spliced to a portion of the target pre-mRNA to from a chimeric mRNA encoding a fusion polypeptide;
• (ii) affinity purifying the fusion polypeptide;
• and
• (iii) determining the peptide sequence of the fusion protein.