Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.
Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Masaki
M
Takeuchi
Yasutaka
Y
Nagaoka
Toshimichi
T
Yamada
Hideo
H
Takakura
Takeaki
T
Ozawa
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D052160:Benzothiazoles / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D001517:Coleoptera / Q000201:enzymology; D000242:Cyclic AMP / Q000378:metabolism*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D057054:Molecular Imaging; D010446:Peptide Fragments / Q000378:metabolism*
D052160:Benzothiazoles; C532924:D-luciferin; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D008156:Luciferases