A FRET-based screening assay for nucleic acid ligands
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- 作者：Daniel Ren?iuk ; Jun Zhou ; Lionel Beaurepaire ; Aurore Gu¨¦din ; Anne Bourdoncle ; Jean-Louis Mergny
- 关键词：Quadruplex ; FRET melting assay ; Unusual nucleic acid structures ; DNA ligands ; Chemical libraries
- 刊名：Methods
- 出版年：2012
- 出版时间：May, 2012
- 年：2012
- 卷：57
- 期：1
- 页码：122-128
- 全文大小：1037 K
文摘
Some of the most serious diseases are characterized by the presence of a specific secondary structure within DNA or RNA, often in the promoter or the coding region of the responsible gene, that enhances or disrupts expression of the protein. Structural elements that impact cellular function may also be formed in other genomic regions such as telomeres. Compounds that interact with such structural elements may be useful in diagnosis or treatment of patients. In this report, we present a FRET melting assay that allows testing of libraries of compounds against four different nucleic acid structures. Compounds are tested to determine whether they stabilize preformed secondary structures (i.e., whether they cause an increase in melting temperature (Tm)). This property is described by the ¦¤Tm parameter, which is the difference between the Tm of the compound-stabilized structure and the Tm of the unbound structure. Model oligonucleotides are labeled with FAM as a fluorescent donor and TAMRA as an acceptor. The intensity of FAM fluorescence is recorded as a function of temperature. Melting temperatures are determined by the FRET method in 96-well plates; this assay could easily be converted into 384-well format.