Ghosn Bilal, Haselton Frederick R, Gee Kyle R, Monroe W Todd
Department of Biological & Agricultural Engineering, Louisiana State University and LSU AgCenter, Baton Rouge, LA 70803, USA.
Photochem Photobiol. 2005 Jul-Aug;81(4):953-9. doi: 10.1562/2004-11-15-RA-373.
Previous reports have shown that 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE-caged oligonucleotides have changed spectrophotometric and electrophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence-based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14-16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light-based technology can be used as a tool for the spatial and temporal regulation of hybridization-based DNA bioactivity.
先前的报告表明，与DNA质粒偶联的1-(4,5-二甲氧基-2-硝基苯基)乙酯(DMNPE)加合物在体外和体内均会阻断转录，直至用光将其去除。在本报告中，我们探索了DMNPE在控制DNA杂交方面的应用。我们发现，DMNPE笼化的寡核苷酸具有改变的分光光度和电泳特性，这些特性可通过光照恢复。笼化的寡核苷酸的电泳迁移率比未笼化的寡核苷酸以及经光照的笼化寡核苷酸慢。使用与30聚体分子信标互补的20聚体笼化DNA寡核苷酸，通过基于荧光的测定法评估了笼化对杂交的影响。荧光结果表明杂交受到抑制，随后通过光照得以恢复。随后的凝胶迁移试验证实了这些结果。每个寡核苷酸平均有14-16个DMNPE加合物的笼化寡核苷酸的杂交活性为未笼化对照寡核苷酸的14%，在365nm光解后，增加到对照的近80%。对经光照然后过滤以去除释放的DMNPE加合物的笼化寡核苷酸进行分光光度表征，结果表明光活化后仍有两到四个附着的笼化基团。这些结果表明，这种基于光的技术可作为一种工具，用于对基于杂交的DNA生物活性进行空间和时间调控。
