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Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay
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DOI：10.1093/nar/gkw117.png)
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Abstract
En 中文
Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop-loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop-loop interactions in hammerhead ribozymes.
Keywords:
MAMMALIAN-CELLS
RNA CIRCUITS
DESIGN
PROTEIN
RIBOSWITCHES
HAMMERHEAD
EXPRESSION
REGULATORS
STRATEGY
BIOLOGY
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