pRNA chimera
a chimera and hammerhead technology, applied in the field of chimeras, can solve the problems of reducing the efficiency of cell cleavage, relatively few reports on the successful use of hammerhead ribozymes in living organisms,
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example 1
Elongation of phi29 pRNA at the 3′ End and Effect on Activity
[0102]To investigate whether additional burdens can be imposed on the pRNA, the 3′ ends of the pRNA were extended with variable lengths.
[0103]The RNA products Eco-pRNA and XbHi-pRNA were produced by in vitro T7 RNA polymerase transcription using DNA templates from plasmid pCRTMII that were precleaved with EcoRI or XbaI / HindIII, respectively. To generate the plasmid pCRTM2, a PCR DNA fragment was produced with the primer pair P7 / P11 to flank the pRNA coding sequence (Zhang et al., Virology 207:442-51 (1995)). The PCR fragment was then cloned into the PCR cloning vector pCRTMII (Invitrogen, Carlsbad, Calif.). DNA sequencing after colony isolation confirmed the presence of the PCR fragment in the plasmid. The RNA product 174-pRNA was either extracted from procapsids, as described by Guo et al. (Science 236:690-94 (1987)) and Wichitwechkarn et al. (Nucl. Acids Res. 17:3459-68 (1989)) or transcribed in vitro with a PCR DNA frag...
example 2
Circularly Permuted φ29 pRNA
[0105]Circularly permitted pRNA (cpRNA) from bacteriophage φ29 was synthesized by way of transcription from a DNA template. The feasibility of constructing circularly permuted RNAs lies in the close proximity of the native φ29 RNA 5′ and 3′ ends (Zhang et al., Virology 201:77-85 (1994)). φ29 pRNA 5′ and 3′ ends are in close proximity. Construction of biologically active circularly permuted pRNAs revealed that interruption of pRNA internal bases did not affect the global folding of pRNA.
[0106]To construct circularly permuted pRNA, two tandem pRNA-coding sequences separated by a 3-base or 17-base loop sequence were cloned into a plasmid (FIG. 11) (see, Zhang et al., Virology 207:442-451 (1995). Plasmids cpDNA3A (1) and cpDNAT7 (II) containing a tandem pRNA coding sequence were connected by 3- or 17-nucleotide synthetic loops, respectively. PCR was used to create dsDNA fragments with non-native 5′ / 3′ ends. In vitro transcription was then performed to generat...
example 3
In Vitro Activity of pRNA-Ribozyme Chimera
[0110]The loop used to connect the native termini of the pRNA in Example 2 did not itself possess any biological activity. However, we wondered whether an RNA sequence with biological activity would retain its activity if tethered at both ends to pRNA. It was decided to test a hammerhead ribozyme as the loop sequence.
[0111]An in vitro model system (FIG. 12) as previously described in Cotton et al. (EMBO J. 8:3861-3866 (1989)) was modified and used as a control to test the functionality of a pRNA-ribozyme chimera. U7snRNA (SEQ ID NO:4) was selected as the target RNA. A chimeric RNA molecule, pRNA-RzU7 (SEQ ID NO:3), was synthesized. This system was used to determine whether the pRNA could harbor other hammerhead ribozymes to function in substrate cleavage (Cotten and Bimstiel, EMBO J 8:3861-3866 (1989)).
[0112]RNAs were prepared as described previously by Zhang et al. (Virology 201:77-85 (1994)). Briefly, DNA oligonucleotides were synthesized ...
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