Reversal of Phospholamban Inhibition of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) Using Short, Protein-interacting RNAs and Oligonucleotide Analogs

Abstract

The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and phospholamban (PLN) complex regulates heart relaxation through its removal of cytosolic Ca²⁺ during diastole. Dysfunction of this complex has been related to many heart disorders and is therefore a key pharmacological target. There are currently no therapeutics that directly target either SERCA or PLN. It has been previously reported that single-stranded DNA binds PLN with strong affinity and relieves inhibition of SERCA in a length-dependent manner. In the current article, we demonstrate that RNAs and single-stranded oligonucleotide analogs, or xeno nucleic acids (XNAs), also bind PLN strongly (K_d <10 nm) and relieve inhibition of SERCA. Affinity for PLN is sequence-independent. Relief of PLN inhibition is length-dependent, allowing SERCA activity to be restored incrementally. The improved in vivo stability of XNAs offers more realistic pharmacological potential than DNA or RNA. We also found that microRNAs (miRNAs) 1 and 21 bind PLN strongly and relieve PLN inhibition of SERCA to a greater extent than a similar length random sequence RNA mixture. This may suggest that miR-1 and miR-21 have evolved to contain distinct sequence elements that are more effective at relieving PLN inhibition than random sequences.

Keywords: Phospholamban (PLN); RNA-protein interaction; calcium ATPase (SERCA); cardiomyopathy; fluorescence anisotropy; microRNA (miRNA); oligonucleotide analogs; protein complex; protein-nucleic acid interaction; structure-function.

FIGURE 1.

RNA affinity for PLN. A, RNA affinity for PLN was measured using ACE and FP. Fluorescently labeled, random sequence RNA libraries (20 nm) at lengths of 5, 10, 20, 35, and 50 were titrated with increasing concentrations of PLN. Error bars are the standard errors from the regression analysis used to estimate K_d (Equation 2, n = 3 replicates measured at 9 PLN concentrations). B, competitive binding assay for PLN between labeled and unlabeled RNA. 100 nm labeled, random sequence, 20-mer RNA + 100 nm PLN was titrated with increasing concentrations of unlabeled, random sequence, 20-mer RNA. Error bars are the standard error of n = 3 replicates. C, example ACE binding curve for random sequence 20-mer RNA. Error bars are the standard errors of n = 3 replicates. D, example FP binding curve for random sequence 20-mer RNA. Error bars are the standard errors of n = 3 replicates.

FIGURE 2.

RNA relieves PLN-mediated inhibition of SERCA. A, normalized SERCA ATPase activity was measured at increasing Ca²⁺ concentrations using a coupled enzyme assay in DOPC:DOPE lipid vesicles. The black line is the activity curve for SERCA alone; the red line is SERCA + PLN, while the other colors represent the addition of different lengths of random sequence RNA to the SERCA + PLN mixture. A regression analysis (Equation 1) was used to estimate pK_Ca (i.e. pCa²⁺ at activity half-maximum) from each curve. Each data point is the average of n = 3 replicates. Error bars were omitted for clarity. B, pK_Ca estimates from A are plotted for different length random sequence RNAs. Error bars are the standard errors from the regression analyses used to estimate pK_Ca (Equation 1, n = 3 replicates measured at 12 Ca²⁺ concentrations). PLN inhibits SERCA by lowering the apparent Ca²⁺ affinity of SERCA, resulting in a lower pK_Ca value. The addition of 50-mer RNA fully restores SERCA activity (no statistical difference from SERCA alone, p > 0.05). All lengths other than the 5-mer and 10-mer are statistically different when compared with WT PLN (p < 0.05). p values were calculated using an unpaired t test.

FIGURE 3.

miR-1 and miR-21 affinity for PLN and functional effect on the PLN/SERCA complex. A, the sequences for miR-1 and miR-21. Secondary structures were predicted using mfold. B, example binding curve of miR-1 to PLN measured using FP. Error bars are the standard errors of n = 3 replicates. C, example binding curve of miR-21 to PLN measured using FP. Error bars are the standard errors of n = 3 replicates. D, dissociation constants for miR-1 and miR-21 for PLN estimated using FP and CE. Error bars are the standard errors obtained from the regression analyses used to estimate K_d values (Equation 2, n = 3 replicates measured at nine PLN concentrations). E, functional effect of miR-1 and miR-21 on the SERCA-PLN complex. Error bars are the standard errors from the regression analyses used to estimate pK_Ca (Equation 1, n = 3 replicates measured at 12 Ca²⁺ concentrations). pK_Ca values after the addition of miR-1 and miR-21 are statistically the same as SERCA alone.

FIGURE 4.

Chemical structures of oligonucleotide analogs (XNAs). Modified XNAs were chosen for their different chemical properties and nuclease resistance. From top to bottom, left to right, the modifications are as follows: d-DNA (naturally occurring DNA bases and backbone), RNA (naturally occurring RNA bases and backbone), PT DNA (replacement of one of the non-bridging oxygens with a sulfur), 2′-O-methyl (2′-O-Me) RNA (the addition of an -OCH₃ group to carbon 2), l-DNA (the enantiomer of natural DNA), PNA (neutral peptide backbone with DNA bases), and morpholino (neutral backbone).

FIGURE 5.

Dissociation (K_d) constants for the XNAs and PLN. K_d values were estimated using FP (black) and ACE (red) for 20-mer random sequence libraries of each oligonucleotide analog. PNA and morpholinos, the two neutral analogs, demonstrated no observable affinity for PLN by either technique. Error bars are the standard errors from the regression analyses used to estimate K_d (Equation 2, n = 3 replicates measured at 9 PLN concentrations).

FIGURE 6.

Oligonucleotide analogs (XNAs) relieve PLN-mediated inhibition of SERCA. A, normalized SERCA ATPase activity was measured at increasing Ca²⁺ concentrations using a coupled enzyme assay in DOPC:DOPE lipid vesicles. The black line is the activity curve for SERCA alone; the red line is SERCA + PLN, while the other colors represent the addition of different 20-mer, random sequence libraries of various oligonucleotide analogs to the SERCA + PLN mixture. A regression analysis (Equation 1) was used to estimate pK_Ca (i.e. pCa²⁺ at activity half-maximum) from each curve. Each data point is the average of n = 6 replicates. Error bars were omitted for clarity. B, pK_Ca estimates from A) are plotted for different oligonucleotide analogs (20-mer, random sequence). Error bars are the standard errors from the regression analyses used to estimate pK_Ca (Equation 1, n = 6 replicates measured at 12 Ca²⁺ concentrations). p values were calculated using an unpaired t test (*, p < 0.05, ***, p < 0.001).