Journal of RNAi and Gene Silencing (October 2005), 1(2), 88-96

Short Report

Design of efficient DNAzymes against muscle AChR a-subunit cRNA in vitro and in HEK 293 cells

Amr Abdelgany ‡, M Khabir Uddin §¥, Matthew Wood ¢, Kazunari Taira §¶ and David Beeson‡*

ABSTRACT

DNAzymes are catalytic DNA which bind to target RNA by complementary sequence arms on a Watson-Crick basis and cleave RNA at specific sites. Potential therapeutic applications require DNAzymes that can efficiently cleave their target. Here we investigate factors affecting DNAzyme cleavage efficacy against the muscle acetylcholine receptor (AChR) a-subunit. The 10-23 DNAzymes cleave at Y-R nucleotide motifs, where R is A or G, and Y is U or C. Targeting a series of sites within different regions of the full-coding length cRNA under simulated physiological conditions found that the most efficient motifs for cleavage were in the hierarchy: GU ≥ AU > GC >>> AC. This order is consistent with the kinetic analysis of short synthetic RNA substrates that have the same binding arms but different cleavage sites. DNAzymes with longer symmetric binding arms were more efficient than those with shorter arms, while asymmetric DNAzymes with a longer arm I were also more efficient, suggesting a dominant role for arm I in determining cleavage activity. Modification of one DNAzyme by inverted thymidine (iT) or locked nucleic acids (LNA) showed the LNA-modified DNAzyme gave efficient silencing of AChR expression in HEK 293 cells. Our data demonstrate the usefulness of screening in vitro for an efficient DNAzyme prior to cellular applications.

KEYWORDS: DNAzymes, RNA cleavage, AChR a -subunit, gene therapy, LNAzymes, gene silencing

‡ Weatherall Institute of Molecular Medicine, University of Oxford, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK.

§ Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan.

¥ Department of Environmental Science, Jahangirnagar University, Dhaka-1342, Bangladesh.

¢ Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK.

¶ Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.

*Correspondence to: David Beeson, Email: dbeeson@hammer.imm.ox.ac.uk, Tel: +44(0)1865 222311 , Fax: +44(0)1865 222402

© Copyright Amr Abdelgany et al

(Received 11 August 2005; Revised 29 September 2005; Accepted 04 October 2005)