Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Patent
1993-12-23
1997-06-24
Rories, Charles C. P.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
435 691, 435 911, 4351723, 4353201, 435 6, 4352523, 435348, 435366, 435419, 435418, 536 231, 536 232, 536 234, 536 241, 536 251, 536 253, C12N 1563, C12N 1579, C12N 510, C12N 121
Patent
active
056416733
DESCRIPTION:
BRIEF SUMMARY
1. Field of the Invention
The present invention is directed to novel trans-splicing ribozymes and methods of cell ablation using these ribozymes.
2. Brief Description of the Background Art
RNA molecules with catalytic activity are called ribozymes or RNA enzymes (Cech, T. R., Ann. Rev. Biochem. 59:543-568 (1990). The Tetrahymena thermophila precursor rRNA contains an intron (a ribozyme) capable of catalyzing its own excision. This ribozyme is one of a class of structurally related Group I introns.
The splicing activity of the modified T. thermophila intron requires the presence of a guanosine cofactor and a divalent cation, either Mg.sup.++ or Mn.sup.++, and occurs via two sequential transesterification reactions (FIG. 1). First, a free guanosine is bound to the ribozyme and its 3' hydroxyl group is positioned to attack the phosphorus atom at the 5' splice site. The guanosine is covalently attached to the intron sequence and the 5' exon is released. Second, the phosphodiester bond located at the 3' splice site undergoes attack from the newly freed 3' hydroxyl group of the 5' exon, resulting in production of the ligated exon sequences. The excised intron subsequently undergoes a series of transesterification reactions, involving its 3' hydroxyl group and internal sequences, resulting in the formation of shortened circular forms.
These successive reactions are chemically similar and appear to occur at a single active site. The reactions of self-splicing are characterized by the formation of alternative RNA structures as differing RNA chains are each brought to form similar conformations around the highly conserved intron. Splicing requires the alignment of the intron-exon junctions across a complementary sequence termed the "internal guide sequence" or IGS.
The first cleavage at the 5' splice site requires the formation of a base-paired helix (P1) between the IGS and sequences adjacent the splice site. The presence of a U:G "wobble" base-pair within this helix defines the phosphodiester bond that will be broken in the catalytic reaction of the ribozyme. After cleavage of this bond, a portion of the P1 helix is displaced and a new helix, P10, is formed due to complementarity between the IGS and sequences adjacent the 3' splice site. An invariant guanosine residue precedes the phosphodiester at the 3' splice site, similar to the portion of the P1 sequence that it is displacing. Thus, ligation of the exons occurs in a reverse of the first cleavage reaction but where new exon sequences have been substituted for those of the intron. It may be noted that intron circularization reactions subsequent to exon ligation also involve base-pairing of 5' sequences across the IGS, and attack mediated by the 3' hydroxyl group of the intron's terminal guanine residue (Been, M. D. et al., "Selection Of Circularizaton Sites In A Group I IVS RNA Requires Multiple Alignments Of An Internal Template-Like Sequence," Cell 50:951 (1987)).
In order to better define the structural and catalytic properties of the Group I introns, exon sequences have been stripped from the "core" of the T. thermophila intron. Cech, T. R. et al., WO 88/04300, describes at least three catalytic activities possessed by the Tetrahymena intron ribozyme: (1) a dephosphorylating activity, capable of removing the 3' terminal phosphate of RNA in a sequence-specific manner, (2) an RNA polymerase activity (nucleotidyl transferase), capable of catalyzing the conversion of oligoribonucleotides to polyribonucleotides, and (3) a sequence-specific endoribonuclease activity.
Isolated ribozyme activities can interact with substrate RNAs in trans, and these interactions characterized. For example, when truncated forms of the intron are incubated with sequences corresponding to the 5' splice junction, the site undergoes guanosien-dependent cleavage in mimicry of the first step in splicing. The substrate and endoribonucleolytic intron RNAs base-pair to form helix P1, and cleavage occurs after a U:G base-pair at the 4th-6th position. Phylogenetic comparisons and mutational an
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Taira,
Brand Andrea
Goodman Howard M.
Haseloff James
Perrimon Norbert
President and Fellows of Harvard College
Rories Charles C. P.
The General Hospital Corporation
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