Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes


Allosteric RNAs operate as molecular switches that alter folding and function in response to ligand binding. A common type of natural allosteric RNAs is the riboswitch; designer RNAs with similar properties can be created by RNA engineering. We describe a computational approach for designing allosteric ribozymes triggered by binding oligonucleotides. Four universal types of RNA switches possessing AND, OR, YES and NOT Boolean logic functions were created in modular form, which allows ligand specificity to be changed without altering the catalytic core of the ribozyme. All computationally designed allosteric ribozymes were synthesized and experimentally tested in vitro. Engineered ribozymes exhibit >1,000-fold activation, demonstrate precise ligand specificity and function in molecular circuits in which the self-cleavage product of one RNA triggers the action of a second. This engineering approach provides a rapid and inexpensive way to create allosteric RNAs for constructing complex molecular circuits, nucleic acid detection systems and gene control elements.

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Figure 1: Design architectures for the construction of oligonucleotide-responsive hammerhead ribozymes.
Figure 2: Design and characterization of an oligonucleotide-specific RNA switch possessing YES logic function.
Figure 3: Design and characterization of YES-2, a variant of YES-1 that exhibits altered oligonucleotide specificity.
Figure 4: Design and characterization of NOT-1 based on an extended hammerhead ribozyme.
Figure 5: Design and characterization of AND-1, an oligonucleotide-specific molecular switch that possesses AND logic function.
Figure 6: Design and characterization of OR-1, an oligonucleotide-specific molecular switch that possesses OR logic function.
Figure 7: A two-step ribozyme signaling pathway constructed using YES-1 and a variant of YES-2.


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We thank members of the Breaker laboratory for helpful discussions. This work was supported by grants from the National Science Foundation and by the Defense Advance Research Projects Agency (DARPA). This project also was supported in part with Federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, under contract No. N01-HV-28186.

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Correspondence to Ronald R Breaker.

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Competing interests

R.R.B. is cofounder of the biotechnology company Archemix (Cambridge, MA, USA) that holds intellectual property on RNA switch technology.

Supplementary information

Supplementary Fig. 1

Design and characterization of YES-3, a variant of YES-1 that exhibits altered oligonucleotide specificity. (PDF 403 kb)

Supplementary Fig. 2

Design and characterization of YES-4, a variant of YES-1 that has altered oligonucleotide specificity. (PDF 398 kb)

Supplementary Fig. 3

Design and characterization of YES-5, a variant of YES-1 that exhibits altered oligonucleotide specificity. (PDF 404 kb)

Supplementary Fig. 4

Design and characterization of YES-6, which is more thermodynamically stable (Ep = −57 kcal mol−1) than the other YES switches. It is design not to cleave even in the presence of corresponding DNA effector. (PDF 160 kb)

Supplementary Fig. 5

Dot plot matrix for NOT-1. See Fig. 3 for details on the sequence and function of NOT-1. (PDF 174 kb)

Supplementary Fig. 6

Dot plot matrix for AND-1. (PDF 282 kb)

Supplementary Fig. 7

Dot plot matrix for OR-1. (PDF 418 kb)

Supplementary Fig. 8

Design and function of three variants of OR-1 that exhibit improved levels of oligonucleotide-triggered self cleavage. (PDF 526 kb)

Supplementary Fig. 9

A flow chart of the computational procedure for the design of RNA switches. (PDF 171 kb)

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Penchovsky, R., Breaker, R. Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes. Nat Biotechnol 23, 1424–1433 (2005).

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