ABSTRACT
The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.
Subject(s)
DNA, Catalytic/radiation effects , Gene Expression Regulation , Gene Expression/radiation effects , Light , Oligodeoxyribonucleotides, Antisense/radiation effects , Base Sequence , Cell Line , DNA/chemistry , DNA/metabolism , DNA, Catalytic/metabolism , Enzyme Activation , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides, Antisense/metabolism , Photochemical Processes , RNA/metabolismABSTRACT
Caged antisense oligodeoxynucleotides (asODNs) are synthesized by linking two ends of linear oligodeoxynucleotides using a photocleavable linker. Two of them (H30 and H40) have hairpin-like structures which show a large difference in thermal stability (Delta T(m) = 17.5 degrees C and 11.6 degrees C) comparing to uncaged ones. The other three (C20, C30 and C40) without stable secondary structures have the middle 20 deoxynucleotides complementary to 40-mer RNA. All caged asODNs have restricted opening which provides control over RNA/asODN interaction. RNase H assay results showed that 40-mer RNA digestion could be photo-modulated 2- to 3-fold upon light-activation with H30, H40, C30 and C40, while with C20, RNA digestion was almost not detectable; however, photo-activation triggered >20-fold increase of RNA digestion. And gel shift assays showed that it needed >0.04 microM H40 and 0.5 microM H30 to completely bind 0.02 microM 40-mer RNA, and for C40 and C30, it needed >0.2 microM and 0.5 microM for 0.02 microM 40-mer RNA binding. However, even 4 microM C20 was not able to fully bind the same concentration of 40-mer RNA. By simple adjustment of ring size of caged asODNs, we could successfully photoregulate their hybridization with mRNA and target RNA hydrolysis by RNase H with light activation.
Subject(s)
Oligodeoxyribonucleotides, Antisense/chemistry , RNA/metabolism , Ultraviolet Rays , DNA, Circular/chemistry , Nucleic Acid Hybridization , Oligodeoxyribonucleotides, Antisense/radiation effects , RNA/chemistry , Ribonuclease H/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation, Neoplastic , Oligodeoxyribonucleotides, Antisense/chemistry , DNA, Neoplasm/analysis , Gene Expression Regulation, Neoplastic/radiation effects , Genes, myb , Humans , K562 Cells , Nucleic Acid Denaturation , Oligodeoxyribonucleotides, Antisense/radiation effects , Photochemistry , RNA, Messenger/metabolism , RNA, Neoplasm/analysis , Ribonuclease H/metabolism , Ultraviolet RaysABSTRACT
Modified DNA carrying an azobenzene was successfully applied to the photo-regulation of DNA/RNA hybridization. When the azobenzene was isomerized from trans- to cis-form on UV-irradiation, the melting temperature of the duplex was significantly lowered. This process was totally reversible so that the Tm increased by cis-->trans isomerization induced by visible light irradiation.
