Sequence Mutation and Structural Alteration Transform a Noncatalytic DNA Sequence into an Efficient RNA-Cleaving DNAzyme

J Mol Evol. 2015 Dec;81(5-6):245-53. doi: 10.1007/s00239-015-9712-x. Epub 2015 Nov 3.


We have previously shown that through test-tube molecular evolution, an arbitrarily chosen noncatalytic DNA sequence can be evolved into a catalytic DNA (DNAzyme) with significant RNA-cleaving activity. In this study, we aim to address the question of whether the catalytic activity of such a DNAzyme can be further optimized using in vitro selection. Several cycles of selective enrichment starting with a partially randomized DNA library have resulted in the isolation of many sequence variations that show notably improved catalytic activity. Bioinformatic analysis and activity examination of several DNAzyme-substrate constructs have led to two interesting findings about sequence mutations and the secondary structure of this DNAzyme: (1) three crucial mutations have transformed the DNAzyme into 8-17, a DNAzyme that has been discovered in multiple previous in vitro selection experiments, and (2) other mutations have allowed this special 8-17 variant to make structural fine-tuning in order to cleave an arbitrarily chosen RNA-containing substrate with a defined sequence. Our study not only showcases the combined power of directed molecular evolution and in vitro selection techniques in turning a noncatalytic nucleic acid sequence into an efficient enzyme, but it also raises the question of whether mother nature has used a similar approach to evolve natural enzymes.

Keywords: 8–17; DNAzyme; In vitro selection; Molecular evolution; RNA cleavage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA, Catalytic / chemistry
  • DNA, Catalytic / metabolism*
  • Kinetics
  • Nucleic Acid Conformation
  • Ribonucleases / metabolism*
  • SELEX Aptamer Technique


  • DNA, Catalytic
  • Ribonucleases