Control of DNA hybridization with photocleavable adducts

被引:42
作者
Ghosn, B
Haselton, FR
Gee, KR
Monroe, WT
机构
[1] Louisiana State Univ, Dept Biol & Agr Engn, Baton Rouge, LA 70803 USA
[2] LSU AgCtr, Baton Rouge, LA 70803 USA
[3] Vanderbilt Univ, Dept Biomed Engn, Nashville, TN USA
[4] Mol Probes Inc, Eugene, OR USA
关键词
D O I
10.1562/2004-11-15-RA-373R1.1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 oligonucleoticle was 14% of noncaged control oligonucleotides and after 365 urn 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.
引用
收藏
页码:953 / 959
页数:7
相关论文
共 41 条
[1]   BIOLOGICALLY USEFUL CHELATORS THAT RELEASE CA-2+ UPON ILLUMINATION [J].
ADAMS, SR ;
KAO, JPY ;
GRYNKIEWICZ, G ;
MINTA, A ;
TSIEN, RY .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1988, 110 (10) :3212-3220
[2]   Photocleavable protecting groups as nucleobase protections allowed the solid-phase synthesis of base-sensitive SATE-prooligonucleotides [J].
Alvarez, K ;
Vasseur, JJ ;
Beltran, T ;
Imbach, JL .
JOURNAL OF ORGANIC CHEMISTRY, 1999, 64 (17) :6319-6328
[3]   Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos [J].
Ando, H ;
Furuta, T ;
Tsien, RY ;
Okamoto, H .
NATURE GENETICS, 2001, 28 (04) :317-325
[4]  
Ando Hideki, 2003, Methods in Cell Science, V25, P25, DOI 10.1023/B:MICS.0000006846.13226.38
[5]   Magnesium precipitate hot start method for PCR [J].
Barnes, WM ;
Rowlyk, KR .
MOLECULAR AND CELLULAR PROBES, 2002, 16 (03) :167-171
[6]   Thermodynamic basis of the enhanced specificity of structured DNA probes [J].
Bonnet, G ;
Tyagi, S ;
Libchaber, A ;
Kramer, FR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (11) :6171-6176
[7]  
Chaulk SG, 2001, ANGEW CHEM INT EDIT, V40, P2149, DOI 10.1002/1521-3773(20010601)40:11<2149::AID-ANIE2149>3.0.CO
[8]  
2-Z
[9]   Caged RNA: photo-control of a ribozyme reaction [J].
Chaulk, SG ;
MacMillan, AM .
NUCLEIC ACIDS RESEARCH, 1998, 26 (13) :3173-3178
[10]   Light-activated gene expression [J].
Cruz, FG ;
Koh, JT ;
Link, KH .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2000, 122 (36) :8777-8778