A molecular beacon-based method allows the detection of ATP, enabling the screening of protein kinase A inhibitors. This method, developed by Ma et al. (2017) is a non-radio isotopic method for assaying protein kinase A.
Protein phosphorylation is a post-translational modification of proteins. Protein kinases catalyze the phosphorylation reaction. Protein phosphorylation plays a significant role in regulating signal transduction, including cell proliferation, hormone secretion, cell differentiation, gene expression, and apoptosis. During the phosphorylation reaction, protein kinases catalyze the addition of a γ-phosphoryl group from adenosine-5’-triphosphate (ATP) to a free hydroxyl group of serine, threonine, or tyrosine in a peptide substrate.
Aberrant protein kinase activities and abnormal protein phosphorylation are the causes of various human diseases.
Ma et al. (2017) quantified ATP levels after a protein kinase reaction using T4 DNA ligase and a molecular beacon, without the need for radioactive labeling.
When ATP is present, DNA ligase catalyzes the ligation of short DNA oligonucleotides. The ligation product can then hybridize to the molecular beacon (MB). The hybridization reaction results in enhanced fluorescence of MB. This assay allows the determination of protein kinase A concentration in the range of 12.5 to 150 nM. The reported detection limit is 1.25 nM. This assay also allows investigation of the effect of genistein on protein kinase A.
Genistein (https://en.wikipedia.org/wiki/Genistein) is a plant-derived aglycone isoflavone that acts as a tyrosine kinase inhibitor, mostly of epidermal growth factor receptors.
Oligonucleotides utilized for the design of the molecular beacon:
MB: 5′-(TAMRA)-CCTCTCCGTGTCTTGTACTTCCCGTCAGAGA GG-(DABCYL)-3′.
The MB is labeled at its 5′-terminal with a fluorescent group, tetramethyl rhodamine (TAMRA). The DNA fragment, N1: 5′-p-TACAAGACAC-3′, is complementary to the 10 bases at the 5′-end of the MB loop needed for 5′-phosphorylation, and fragment N2: 5′-GACGGGAAG-3′ is complementary to the 9 bases at the 5′ end of the MB loop.
Illustration of the protein kinase A activity assay.
[1] Kemptide (LRRAS(OH)LG) + ATP -> LRRAS(OPO3H2)LG in the presence of the protein kinase. Protein kinase A catalyzes the phosphorylation of kemptide.
[2] The detection of ATP using molecular beacons (MBs) is based on DNA ligation. In the presence of ATP, the T4 DNA ligase catalyzes the ligation of 2 short oligonucleotides, forming a longer oligonucleotide. The ligation product hybridizes with the MB and restores its fluorescence.
Ma et al. observed that the rate of fluorescence enhancement was significantly lower whenever ATP was partially consumed, showing that this method allows the detection of protein kinase A activity.
Reference
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Ma C, Lv X, Wang K, Jin S, Liu H, Wu K, Zeng W. Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe. Bioengineered. 2017 Nov 2;8(6):716-722. [PMC]
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