Figure 1

αDENV-GrpI targeting and trans -splicing of DENV. Schematic diagrams showing DENV sequences targeted by 9 series (A) and 96 series (B) αDENV-GrpIs (shown as blow-ups), and the trans-splicing reaction mediated by these introns (C) as previously described for other trans-splicing ribozymes (5). A) A region of the DENV genome targeted by the 9 series intron is conserved among all serotypes based on Clustal X DENV genome alignment of all 98 DENV sequences deposited at GenBank. All accession numbers are listed in Methods. Shaded areas indicate 100% conserved bases. The arrows designate U residues targeted for cleavage. The residues contributing to structural elements of the intron RNA-target RNA complexes in the different 9 series introns are as follows: Internal Guide Sequence (IGS) = U138 to A146, Bulge Loop (BL) = A147 to A 152, and External Guide Sequence (EGS) = G153 to G161 as indicated on. Uracil 143 is targeted for cleavage. The single adenine at base 152 is not fully conserved and has therefore been engineered into LB, which is not required to base pair with the target for efficient splicing. B) Primary sequence of the DENV 2-NGC genome depicting the residues contributing to structural elements of the intron RNA-target RNA complexes in the different 96 series intron structures: IGS = A127 to C135, LB = A136 to A 139, and EGS = U140 to U235. The uracil at position 132 is targeted for cleavage. C) The αDENV-GrpI- mediated trans-splicing reaction. The DENV target sequence is represented as the upper schematic diagram with the trans-splicing ribozymes shown below. See text for description. Figure not drawn to scale.