In vitro selection and characterization of DNA aptamers specific for phospholamban

Calcium transport across the membrane of the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart muscle contraction and relaxation. The sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2a is responsible for Ca(2+) up-take by this organelle and is inhibited in a reversible manner by phospholamban, another SR membrane protein. Thus, alleviation of phospholamban-mediated inhibition of SERCA2a is a potential therapeutic option for heart failure and cardiomyopathy. We have now applied the systematic evolution of ligands by exponential enrichment protocol to a library of single-stranded DNA molecules containing a randomized 40-nucleotide sequence to isolate aptamers that bind phospholamban. One of the obtained aptamers, designated Apt-9, was found to specifically bind to the cytoplasmic region of phospholamban in vitro with high affinity (dissociation constant, approximately 20 nM). Apt-9 increased the Ca(2+)-dependent ATPase activity of cardiac SR vesicles but not that of SR vesicles from skeletal muscle in a concentration-dependent manner. It also shifted the Ca(2+) concentration-response curve for this ATPase activity to the left. These effects of Apt-9 were not mimicked by an oligonucleotide with a scrambled version of the Apt-9 sequence. Thus, our results indicate that Apt-9 activates SERCA2a by alleviating the inhibitory effect of phospholamban on this ATPase, and they suggest that phospholamban-specific aptamers warrant further investigation as potential therapeutic agents for heart failure and cardiomyopathy.