Puromycin dihydrochloride

Puromycin dihydrochloride [1] is an analog of aminoacyl-tRNA. Puromycin dihydrochloride allows selection for cells expressing the resistance gene puromycin N-acetyl-transferase (PAC) and is useful in CRISPR/Cas9 mammalian gene editing by selecting for successful Cas9-induced knock-in with puromycin resistance gene. It inhibits the incorporation of aminoacyl-tRNA into the C-terminal on a synthesizing polypeptide, resulting in the premature termination of the polypeptide chain.

Puromycin is toxic to the growth of various eukaryote cells including mammalian cells. Concentrations of puromycin sufficient to inhibit the cell growth of mammalian cells range from 0.5-10 μg/ml. IC90s for puromycin to inhibit the growth of Plasmodium falciparum and Giardia lamblia are 60 ng/ml and 54 μg/ml, respectively [2]. Aminoacyl-tRNA is used as the material for elongation of a synthesizing polypeptide on a ribosome [2].

When treated with puromycin dihydrochloride at different concentrations, the growth rates of T. thermophila changed. In the first 24 h, puromycin dihydrochloride at a concentration of 50 µg/ml reduced the growth rate by 80%, but did not completely block the cell growth; until 72 h, there was a gradual cell number increase. At 100 μg/ml, puromycin dihydrochloride completely blocked the cell growth; in the first 48 h under this condition, almost all of the cells died, surviving cells grew rapidly after 48 h. Puromycin dihydrochloride at 150 μg/ml completely inhibited the cell growth for 72 h. By 72 h, the majority of cells died, and then surviving cells grew. Puromycin dihydrochloride at 200 μg/ml made almost all the cells die by 48 h, and hence no survivors appeared [2].

In animals of 25 days old, 180 or 120 min of previous exposure to puromycin dihydrochloride inhibited subsequent amino acid transport. In animals of 50 days old, however, puromycin dihydrochloride failed to inhibit α-aminoisobutyric acid uptake [3].

References:
[1].  Mi. Sundaralingami and S. K. Arora. Stereochemistry of nucleic acids and their constituents. IX. The conformation of the antibiotic puromycin dihydrochloride pentahydrate. Proc. N. A. S., 1969, 64(3):1021-6.
[2].  Masaaki Iwamoto, ChieMori, Yasushi Hiraoka, et al. Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena. Gene, 2014, 534:249–255.
[3].  Elsas L.J. II, MacDonell R.C. Jr. and Rosenberg L.E. Influence of age on insulin stimulation of amino acid uptake in rat diaphragm. J. Biol. Chem., 1971, 246(21):6452-6459.