An Efficient Catalytic DNA that Cleaves L-RNA

PLoS One. 2015 May 6;10(5):e0126402. doi: 10.1371/journal.pone.0126402. eCollection 2015.

Abstract

Many DNAzymes have been isolated from synthetic DNA pools to cleave natural RNA (D-RNA) substrates and some have been utilized for the design of aptazyme biosensors for bioanalytical applications. Even though these biosensors perform well in simple sample matrices, they do not function effectively in complex biological samples due to ubiquitous RNases that can efficiently cleave D-RNA substrates. To overcome this issue, we set out to develop DNAzymes that cleave L-RNA, the enantiomer of D-RNA, which is known to be completely resistant to RNases. Through in vitro selection we isolated three L-RNA-cleaving DNAzymes from a random-sequence DNA pool. The most active DNAzyme exhibits a catalytic rate constant ~3 min-1 and has a structure that contains a kissing loop, a structural motif that has never been observed with D-RNA-cleaving DNAzymes. Furthermore we have used this DNAzyme and a well-known ATP-binding DNA aptamer to construct an aptazyme sensor and demonstrated that this biosensor can achieve ATP detection in biological samples that contain RNases. The current work lays the foundation for exploring RNA-cleaving DNAzymes for engineering biosensors that are compatible with complex biological samples.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Aptamers, Nucleotide / metabolism
  • Base Sequence
  • Biocatalysis
  • Biosensing Techniques*
  • DNA, Catalytic / metabolism*
  • Nucleic Acid Conformation
  • RNA / metabolism*

Substances

  • Aptamers, Nucleotide
  • DNA, Catalytic
  • RNA
  • Adenosine Triphosphate

Grant support

The research work was supported by the Natural Sciences and EngineeringResearch Council of Canada Discovery Grant (227594-2009) and by a research grant to YL from the Sentinel Bioactive Paper Network. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.