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Nucleic Acids Research Advance Access originally published online on December 1, 2007
Nucleic Acids Research 2008 36(2):559-569; doi:10.1093/nar/gkm1029
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Nucleic Acids Research, 2008, Vol. 36, No. 2 559-569
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides

XinJing Tang1, Jyothishmathi Swaminathan2, Alan M. Gewirtz2 and Ivan J. Dmochowski1,*

1The Department of Chemistry and 2Division of Hematology/Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

*To whom correspondence should be addressed. Tel: 215-898-6459; Fax: 215-898-2037; Email: ivandmo{at}sas.upenn.edu

Received August 18, 2007. Revised October 20, 2007. Accepted October 29, 2007.

Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the ‘caged’ state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (Tm) upon UV irradiation ({Delta}Tm = –29°C). The most thermally stable conjugate, C6 (Tm = 84°C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex.


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