Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.
An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms.
Nakagawa
Kazuhiro
K
Life Science Laboratory, Advanced Materials Laboratories, Sony Corporation, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8510, Japan. kazuhiro.nakagawa@jp.sony.com
Yamamoto
Takuro
T
Yasuda
Akio
A
D056930:ARNTL Transcription Factors / Q000032:analysis*; D001080:Apurinic Acid / Q000378:metabolism; D056926:CLOCK Proteins / Q000032:analysis*; D002940:Circadian Rhythm; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015341:Nucleic Acid Probes / Q000737:chemistry*; D011119:Polynucleotides / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference
D056930:ARNTL Transcription Factors; D015341:Nucleic Acid Probes; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; D056926:CLOCK Proteins