Nucleic Acids Research, 2003, Vol. 31, No. 20 5982-5992
© 2003 Oxford University Press
RNA cleaving ‘10-23’ DNAzymes with enhanced stability and activity
Free University Berlin, Institute of Chemistry (Biochemistry), Thielallee 63, D-14195 Berlin, Germany and 1 Free University Berlin, Institute for Infectious Diseases Medicine, University Hospital Benjamin Franklin, Hindenburgdamm 27, D-12203, Berlin, Germany
*To whom correspondence should be addressed. Tel: +49 30 83856969; Fax: +49 30 83856413; Email: jkurreck{at}chemie.fu-berlin.de
‘10-23’ DNAzymes can be used to cleave any target RNA in a sequence-specific manner. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides without obstructing cleavage activity. In this study, we optimise the design of a DNAzyme targeting the 5'-non-translated region of the human rhinovirus 14, a common cold virus, with regard to its kinetic properties and its stability against nucleases. We compare a large number of DNAzymes against the same target site that are stabilised by the use of a 3'-3'-inverted thymidine, phosphorothioate linkages, 2'-O-methyl RNA and locked nucleic acids, respectively. Both cleavage activity and nuclease stability were significantly enhanced by optimisation of arm length and content of modified nucleotides. Furthermore, we introduced modified nucleotides into the catalytic core to enhance stability against endonucleolytic degradation without abolishing catalytic activity. Our findings enabled us to establish a design for DNAzymes containing nucleotide modifications both in the binding arms and in the catalytic core, yielding a species with up to 10-fold enhanced activity and significantly elevated stability against nucleolytic cleavage. When transferring the design to a DNAzyme against a different target, only a slight modification was necessary to retain activity.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. R. Dass, P. F.M. Choong, and L. M. Khachigian DNAzyme technology and cancer therapy: cleave and let die Mol. Cancer Ther., February 1, 2008; 7(2): 243 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Donini, M. Clerici, J. Wengel, B. Vester, and A. Peracchi The Advantages of Being Locked: ASSESSING THE CLEAVAGE OF SHORT AND LONG RNAs BY LOCKED NUCLEIC ACID-CONTAINING 8 17 DEOXYRIBOZYMES J. Biol. Chem., December 7, 2007; 282(49): 35510 - 35518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bhindi, R. G. Fahmy, H. C. Lowe, C. N. Chesterman, C. R. Dass, M. J. Cairns, E. G. Saravolac, L.-Q. Sun, and L. M. Khachigian Brothers in Arms: DNA Enzymes, Short Interfering RNA, and the Emerging Wave of Small-Molecule Nucleic Acid-Based Gene-Silencing Strategies Am. J. Pathol., October 1, 2007; 171(4): 1079 - 1088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Silverman In vitro selection, characterization, and application of deoxyribozymes that cleave RNA Nucleic Acids Res., November 11, 2005; 33(19): 6151 - 6163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Schmidt, S. Borkowski, J. Kurreck, A. W. Stephens, R. Bald, M. Hecht, M. Friebe, L. Dinkelborg, and V. A. Erdmann Application of locked nucleic acids to improve aptamer in vivo stability and targeting function Nucleic Acids Res., October 27, 2004; 32(19): 5757 - 5765. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Zhang, C. R. Dass, E. Sumithran, N. Di Girolamo, L.-Q. Sun, and L. M. Khachigian Effect of Deoxyribozymes Targeting c-Jun on Solid Tumor Growth and Angiogenesis in Rodents J Natl Cancer Inst, May 5, 2004; 96(9): 683 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Fahmy and L. M. Khachigian Locked nucleic acid modified DNA enzymes targeting early growth response-1 inhibit human vascular smooth muscle cell growth Nucleic Acids Res., April 23, 2004; 32(7): 2281 - 2285. [Abstract] [Full Text] [PDF]
|
|