Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides.
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 (T(m)) upon UV irradiation (DeltaT(m) = -29 degrees C). The most thermally stable conjugate, C6 (T(m) = 84 degrees 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.
The Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
XinJing
X
Tang
Jyothishmathi
J
Swaminathan
Alan M
AM
Gewirtz
Ivan J
IJ
Dmochowski
D004273:DNA, Neoplasm / Q000032:analysis; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D020570:Genes, myb; D006801:Humans; D020014:K562 Cells; D009691:Nucleic Acid Denaturation; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry* / Q000528:radiation effects; D010777:Photochemistry; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D016914:Ribonuclease H / Q000378:metabolism; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016914:Ribonuclease H