Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes

Garrett Soukup, Elizabeth C. Derose, Makoto Koizumi, Ronald R. Breaker

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3′,5′-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.

Original languageEnglish
Pages (from-to)524-536
Number of pages13
JournalRNA
Volume7
Issue number4
DOIs
StatePublished - 2001

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Catalytic RNA
RNA
Ligands
Cyclic Nucleotides
Mutagenesis
Structural Models
Biosensing Techniques
Phylogeny
Nucleic Acids
Catalytic Domain
Nucleotides

All Science Journal Classification (ASJC) codes

  • Genetics
  • Molecular Biology

Cite this

Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes. / Soukup, Garrett; Derose, Elizabeth C.; Koizumi, Makoto; Breaker, Ronald R.

In: RNA, Vol. 7, No. 4, 2001, p. 524-536.

Research output: Contribution to journalArticle

Soukup, Garrett ; Derose, Elizabeth C. ; Koizumi, Makoto ; Breaker, Ronald R. / Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes. In: RNA. 2001 ; Vol. 7, No. 4. pp. 524-536.
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