Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides

The membrane protein complex between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN’s inhibitory effects on SERCA, increasing the ATPase’s apparent Ca2+ affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca2+ flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.

it when phosphorylated at Ser16 by protein kinase A 6 . Aberrant interactions between SERCA and PLN mutants and concomitant Ca 2+ mishandling have been correlated with dysfunctional contractility and heart disease 7,8 . As a result, several approaches have been pursued to reverse these conditions; not only SERCA gene transfer therapy 4,9,10 , but also siRNA 11 , miRNA inhibition 12 and aptamers 13,14 , have shown promise as therapeutic avenues. In particular, SERCA-directed gene therapy is the most effective strategy to augment Ca 2+ transport and muscle contractility, either using an adeno-associated virus to overexpress the ATPase in cardiomyocytes or targeting PLN to reverse SERCA inhibition 10 . While the former approach is being evaluated in clinical trials, the latter presents challenges. In fact, PLN-knockout mice 15 and PLN-null mutations in humans 16 progress to heart failure and lethal dilated cardiomyopathy, respectively. Although encouraging for large animal models 17 , gene transfer of PLN S16E , a pseudo-phosphorylated mutant, hampers β -adrenergic control of heart contractility as the mutant can no longer be phosphorylated. In addition, current gene therapy methods do not allow a controllable response to counteract the different degrees of heart disease manifestations 4 . Whereas phosphorylatable mutants mimicking the phosphorylated state of PLN are being developed 18,19 , there is a critical need to devise more direct, viable methods to target PLN.
Here, we report the unexpected discovery that small RNAs and single-stranded DNAs (ssDNAs) bind with low nanomolar dissociation constants (K d ) to the SERCA/PLN complex, regulating the ATPase's apparent Ca 2+ affinity in a tunable manner. Specifically, we found that both short RNAs and ssDNAs are able to reverse PLN's inhibitory effects irrespective of their primary sequence. The functional effects are tunable by increasing or decreasing the oligonucleotides' lengths, becoming significant for sizes encompassing typical endogenous miRNAs and reaching a plateau at 80 bases. Solid-state NMR and fluorescence spectroscopy data show that ssDNA binds PLN's cytoplasmic domain specifically, but does not affect SERCA in the absence of the regulatory protein. In particular, NMR spectra show that ssDNA shifts the conformational equilibrium of the SERCA/PLN complex from inhibitory to a non-inhibitory state. Förster resonance energy transfer (FRET) experiments in HEK cells overexpressing the SERCA/ PLN complex show that PLN remains bound to SERCA upon interacting with ssDNA. These functional effects, tested in membrane reconstituted systems, are reproducible in mammalian sarcoplasmic reticulum (SR) cardiac preparations. These findings indicate that chemically modified, non-coding RNA and single-stranded DNA templates with low off-target propensity can be exploited for developing compounds to target SERCA inhibition by PLN, thereby regulating Ca 2+ transport in the SR. The rheostatic control of SERCA function achieved here opens up new possibilities for devising a graded response to varying extents of cardiac pathologies 10 .

Results
Small RNAs and ssDNAs bind PLN with low nanomolar dissociation constants, reversing its inhibitory effect on SERCA. Using affinity capillary electrophoresis (ACE), fluorescence polarization (FP), and native gel mobility shift assays, we found that short RNAs, of similar length to naturally occurring miRNA sequences, display low nanomolar dissociation constants for PLN. As an example, we report the dissociation constant of a random sequence RNA (50mer) to PLN in Fig. 1A and typical binding curves (80mer) in Fig. S1. Importantly, we found that these short RNAs not only bind, but also reverse PLN's inhibition of SERCA. Fig. 1B shows the effect of the 50mer RNA on SERCA activity as monitored using coupled enzyme assays. The Ca 2+ concentration at half maximal activity in the coupled assay curves indicates SERCA's apparent affinity for Ca 2+ ions (pK Ca ). When SERCA is bound to PLN WT , the ATPase activity decreases and the normalized curves show a concomitant reduction of the pK Ca value. Upon binding the 50mer RNA, SERCA's function is nearly restored (Fig. 1B), mimicking the effect of PLN phosphorylation at Ser16 20 . Since aptamers have been selected to target specific proteins without off-target effects on gene expression 21 , we tested the effects of a 50mer ssDNA random sequence. Indeed, we found that both short RNAs and ssDNAs have similar K d values for PLN (Fig. 1A) and restoration of SERCA function (Fig. 1B).
We also assessed the affinity of different lengths of ssDNAs with sequences chosen at random that had no significant secondary structures, with the exception of the 30mer (Table 1 and Fig. 1C,D). We found sequence length to be the best indicator of affinity. Any sequence containing more than 10 bases had low nanomolar K d values, while sequences containing 10 bases or less had significantly less affinity with higher K d values. Surprisingly, all of the randomly chosen sequences containing more than 10 bases exhibited high affinity for PLN, suggesting that this is a sequence-independent interaction ( Table 1). The 80mer used for the binding studies was synthesized as a completely randomized mixture, with a 25% probability of each base being present at every register position. This random library allowed affinity to be determined independently of a specific sequence. The high affinity (low nanomolar K d ) between the completely randomized sequence mixture of 80mer ssDNA and PLN confirms the sequence independence of this interaction. Among the longer sequences, the 30mer demonstrated slightly weaker affinity for PLN than predicted based on sequence length alone. This 30mer is the only sequence listed in Table  1 that exhibits significant secondary structure. The weaker than predicted affinity suggests a thermodynamic penalty to unfold the ssDNA before binding PLN. To analyze the nature of the intermolecular interactions, we carried out the binding assays at different salt concentrations (Fig. S2). Indeed, we found Scientific RepoRts | 5:13000 | DOi: 10.1038/srep13000  Table 1.  that the ssDNA/PLN interactions are persistent even at high salt concentrations and remain in the nanomolar range at NaCl concentrations up to 200 mM. We then performed ATPase assays with varying length oligonucleotides and found they give rise to a graded effect on ATPase activity. Figure 2A depicts the normalized SERCA/PLN activity curves upon addition of ssDNA at different lengths and varying concentrations of free calcium (pCa). In the absence of ssDNA, PLN binding results in decreased Ca 2+ affinity of SERCA (lowest pK Ca , with the brown curve shifted furthest to the right in Fig. 2A). Addition of ssDNA to the PLN/SERCA complex shifts the activity curve toward the higher pCa, indicating that the SERCA's apparent affinity for Ca 2+ ions is increasing; ssDNA reverses the inhibitory effect of PLN ( Fig. 2A,B). The functional effect trends with sequence length, but not necessarily according to the relative affinity of the oligonucleotides (K d values). This is evidenced by the 30mer, which displays a higher K d than expected based on length alone, but still follows the length trend seen in the activity assays ( Fig. 2A,B). It should be noted that all ATPase assays were performed at saturating ssDNA concentrations. Thus, the length trend ( Fig. 2) observed is structural in origin and independent of the ssDNA concentration. Remarkably, longer oligonucleotides result in complete inhibition relief, an effect that mimics phosphorylation of PLN at Ser16 by protein kinase A 22 . With oligonucleotide sequences longer than 50 bases, we observed no further increase of SERCA activity beyond the physiological window, indicating that the effect is mediated by the direct interaction between ssDNA and PLN.
To confirm the specificity of the ssDNA/PLN interactions, we incubated 80mer ssDNA with SERCA in the absence of PLN. Under these conditions, the apparent Ca 2+ affinity was identical to that of SERCA alone, confirming that ssDNA does not have an activating effect without PLN (Fig. 2B). Additionally, both double-stranded DNA and a mixture of free deoxynucleotides (dNTP) show no effect on PLN's inhibition of SERCA; the pK Ca with either double-stranded DNA or free dNTPs is identical to the pK Ca of the PLN inhibited pump (Fig. S3). Taken together, these data suggest that reversal of the inhibitory effect of ssDNAs is manifested in a length-dependent manner, with inhibition relief being progressively more effective with increasing sequence length and reaching a plateau at ~80 oligonucleotides. ssDNA disrupts PLN inhibition without dissociating the SERCA/PLN complex. To probe the specific interactions between PLN and ssDNA both in the absence and presence of SERCA, we labeled  (Table 1) to the pre-formed SERCA/PLN complex under conditions similar to the activity assays did not change the FRET signal. These measurements were repeated with excess PLN, or excess ssDNA, and the results were similar to the FRET data with stoichiometrical amounts (Fig. 3B,C). The enhanced FRET and shift of the emission maximum seen with a 10-fold excess of PLN is indicative of more FRET between SERCA and PLN, resulting from saturating the PLN binding site in the ATPase (Fig. 3C). We have also recorded fluorescence of SERCA AEDANS without PLN, but in the presence of ssDNA. The fluorescence intensity was identical to that of SERCA AEDANS , illustrating that ssDNA does not interact with the ATPase alone (Fig. 3A). The FRET, ACE and fluorescence polarization data collectively demonstrate that the ssDNA interacts specifically with PLN irrespective of the presence of SERCA. Furthermore, ssDNA relieves PLN inhibition by causing a structural rearrangement of PLN without dissociating it from SERCA, in a manner resembling PLN phosphorylation at Ser16 -the endogenous mechanism for inhibition relief.
ssDNA interacts with the SERCA/PLN complex in living cells. The SERCA/PLN interaction studies were also carried out in HEK cells. Specifically, FRET in-cell assays were used to investigate the interactions of ssDNA with free PLN and in complex with SERCA 23,24 . We utilized PLN AFA , a pentamer-destabilizing mutant with full inhibitory activity to minimize PLN oligomerization and detect small changes in binding affinity. Although it runs as a monomer on SDS-PAGE, PLN AFA has a slight tendency to form pentamers in membranes and exhibits a significant intra-pentameric FRET when overexpressed in living cells. A cell-by-cell comparison of FRET with PLN expression level revealed a hyperbolic dependence of FRET efficiency on protein concentration for a population of HEK cells expressing the fluorescent proteins Cer-PLN AFA and YFP-PLN AFA , fused at the N-terminus of PLN (Fig.  S4). From this curve, we calculated the apparent dissociation constant for oligomerization (K D 1) as well as the intrinsic FRET efficiency of the PLN oligomer (FRET max ), reporting on the inter-protomer binding affinity and quaternary structure changes, respectively. In the absence of ssDNA, we found that PLN AFA is able to form pentamers, though with reduced propensity compared to PLN WT . In contrast, addition of the 50-mer ssDNA to the cells containing plasmids encoding for Cer-PLN AFA and YFP-PLN AFA induced a notable increase in the apparent PLN-PLN affinity, representing an approximately 20% increase in PLN oligomerization (Fig. S4). A significant decrease in FRET max was observed, which corresponds to an increase of the donor-acceptor distance (i.e., increased interprotomer affinity) as previously observed for PLN R9C and its phosphomimetic mutants 25,26 . For the SERCA/PLN complex, cell transfection with 50-mer ssDNA had striking effects both on the binding affinity and the structure of the complex. Unlike the in vitro fluorescence experiments in which the AEDANS donor probe was attached at Cys674 in the SERCA's P-domain, the Cer probe placed on the A-domain of SERCA is able to detect a four-fold increase of PLN affinity for SERCA upon oligonucleotide addition, with a concomitant decrease of SERCA/PLN FRET max (Fig. 4). Taken with the in vitro ATPase activity assays, these data demonstrate that ssDNA binding to PLN mimics both the structural and functional effects of phosphorylation on the SERCA/ PLN regulatory complex. The decrease of FRET max upon complex formation is suggestive of a structural rearrangement of PLN within the complex; rather than the dissociation of PLN from the ATPase.

ssDNA reverses PLN's inhibition of SERCA in cardiac SR preparations.
To assess the reproducibility of the ssDNA effects observed in the reconstituted systems in more biologically relevant conditions, we performed the activity assays using crude cardiac SR preparations isolated from pig ventricles, containing the SERCA2a isoform of the ATPase (see Material and Methods). Since PLN is endogenously expressed in ventricles, direct addition of ssDNA to the heavy SR preparations containing the SERCA2a/ PLN complex would be expected to increase the ATPase apparent Ca 2+ affinity. Indeed, addition of ssDNA (1 μ M 80mer) to the pig crude SR vesicles augmented the pK Ca by 0.15 (Fig. 2C). While the effect of ssDNA on SERCA activity in pig crude SR is somewhat lower than in the reconstituted system; this slight discrepancy is likely due to the challenges in determining the exact amounts of the SERCA/PLN complex in these native preparations, as well as their molar ratio and phosphorylation state. Overall, these data confirm the efficacy of ssDNA in relieving PLN inhibition of the mammalian SERCA2a/PLN complex under native conditions. NMR mapping of ssDNA binding epitope on PLN. To identify the specific residues of PLN interacting with the ssDNA, we used solid-state NMR (ssNMR) spectroscopy. We reconstituted U-13 C/ 15 N labeled PLN in deuterated DMPC lipid vesicles and monitored the chemical shifts of the backbone and side chain 13 C resonances in the presence and absence of ssDNA (80mer). To detect the dynamic cytoplasmic domain of PLN, we used the refocused [ 1 H, 13 C]-RINEPT experiment 27 , which is well suited for protein domains undergoing fast reorientation (cytoplasmic domain of PLN) and insensitive to rigid domains on the NMR time scale (PLN's transmembrane domain) 28 . In the free form of PLN, the resonances corresponding to the cytoplasmic region are all detectable. Addition of ssDNA to PLN at a 1:1 molar ratio causes the intensities of several amino acids peaks in the [ 1 H, 13 C]-RINEPT spectrum to decrease, with several peaks becoming broadened beyond detection (Fig. 5). The latter indicates the rigidification of PLN's cytoplasmic domain and an increase in rotational correlation time upon ssDNA binding. In contrast, natural abundance 13 C lipid signals are not affected by ssDNA and remain essentially unchanged (Fig. 5) 29 , but provides visualization of the cytoplasmic domain resonances. Addition of 15mer ssDNA causes extensive line broadening (residues Glu2, Thr8, Ala11, Ile12/18 Arg13/14, and Met20 - Fig. S5), which is in qualitative agreement with ssNMR experiments. Taken with the FRET data, the NMR experiments indicate that the transmembrane domain of PLN remains essentially unperturbed and attached to the ATPase, while ssDNA primarily targets the cytoplasmic domain of PLN interfering with its regulatory function of SERCA.
Previously, we found that PLN bound to SERCA undergoes conformational transitions between three major states (Fig. 6b): an inhibitory T state, with the transmembrane (TM) domain bound to SERCA and the cytoplasmic domain associated to the membrane; an inhibitory R state, with the TM domain bound to SERCA and the cytoplasmic domain unfolded and dissociated from the membrane;   perturbations upon the addition of ssDNA, indicating binding between ssDNA and PLN AFA (Fig. S6). Dipolar assisted rotational resonance (DARR) MAS [ 13 C, 13 C] experiments of U-13 C/ 15 N labeled PLN in complex with unlabeled SERCA were performed in the absence and presence of ssDNA (Fig. 6). Specifically, the DARR peaks corresponding to the Cα /Cβ correlations of the two Ala residues are sensitive to the conformational equilibrium between the different states and can be used as reporter residues (Fig. 6). In the absence of SERCA at 20 °C, these two residues populate a major conformational state (T state) and a sparsely populated R state. Upon addition of SERCA, there is a population shift toward the bound state that is augmented upon phosphorylation of PLN. Upon binding ssDNA, we observe a progressive shift of the two Ala residues of PLN AFA toward lower fields, similar to what is observed with the non-inhibitory phosphorylated form of PLN. These results strongly support the fluorescence studies and show that ssDNA does not detach PLN from SERCA; rather it shifts the conformational equilibrium toward a non-inhibitory or D (DNA bound) state in a manner similar to the phosphorylated state. A hypothetical model of the conformational equilibrium of the SERCA/PLN complex and the effects of ssDNA is reported in Fig. 6B.

Discussion
Homeostatic regulation of Ca 2+ ions in muscle is crucial to proper contractility 5 . SERCA plays a central role in this delicate equilibrium and its function is regulated within a physiological window by PLN 28 . Super-inhibition of SERCA by PLN mutants or hyper-phosphorylation of PLN tip the homeostatic balance and lead to cardiomyopathies 2,30 . So far, there have been no drugs developed with a direct effect on the SERCA/PLN complex. However, the complexity of Ca 2+ transport dysregulation in heart failure calls for therapeutic approaches that parallel the extent of the pathology, which is yet to be achieved.
Our study demonstrates that short RNAs and ssDNAs are able to regulate SERCA activity in a tunable manner by binding the cytoplasmic domain of PLN with nanomolar dissociation constants. Binding of ssDNA to the SERCA/PLN complex induces structural changes and promotes inhibition relief. Due to the high affinity of ssDNA for PLN, the tunability of the effects is not concentration-dependent, but rather length-dependent, with a range from 5 nucleotides, exhibiting minimal effects on SERCA activity, to 80 nucleotides, conferring a nearly complete relief of inhibition. The binding of the ssDNA is likely driven by the electrostatic interactions between the negatively charged phosphate groups of the oligonucleotide and the positively charged Lys and Arg side-chains of PLN. Such charge-charge interactions have been found in sequence-independent ssDNA binding proteins, such as T4 gp32, adeno-DBP and multiple others [31][32][33] . However, electrostatic interactions alone do not fully justify the unique binding seen between ssDNA and PLN. Single chain nucleic acids are essential for PLN binding, since neither double-stranded DNA nor a mixture of free dNTPs are able to reverse PLN inhibition of SERCA. ssDNA binds PLN without detaching it from the ATPase; rather, it shifts its conformational equilibrium toward a non-inhibitory state 18,28 . Previously, we found that PLN bound to SERCA undergoes conformational transitions between three major states: an inhibitory T state, with the transmembrane (TM) domain bound to SERCA and the cytoplasmic domain associated to the membrane; an inhibitory R state, with the TM domain bound to SERCA and the cytoplasmic domain unfolded and dissociated from the membrane; and a sparsely populated, non-inhibitory B or bound state, with both the TM and cytoplasmic domains interacting with SERCA 28 . By tracing the population of the SERCA-bound state of PLN, we found its conformational equilibrium is driven by ssDNA toward a new, distinct state (D state) (Fig. 6), which is non-inhibitory. Since FRET experiments show no detachments of PLN from SERCA, we surmise that the ssDNA may adopt a PLN-bound conformation with the phosphate backbone mimicking the effects of Ser16 and/or Thr17 phosphorylation and restoring SERCA's apparent affinity for Ca 2+ ions.
An important corollary to our study is the possibility of direct interactions of endogenous short RNA sequences (such as miRNA) with the SERCA/PLN complex. In the heart, miRNAs are involved both in cardiogenesis and disease including: hypertrophy, ischemia, and electrical remodeling [34][35][36][37] . miRNAs are up-or down-regulated in response to stress during cardiac disorders 37 and they are notorious for their pleiotropic action, affecting multiple genes in different tissues 37 . The latter has prevented the progression of miRNA regulation to therapeutic applications 38,39 . Based on the high, non-sequence specific affinity of short RNAs for PLN, it is possible that endogenous miRNAs may have a role in cardiac pathophysiology that goes beyond gene regulation. As miRNAs of different lengths and sequences are produced for both protective and pathological roles 34,36,40 , the up-regulation of miRNAs under cardiac remodeling and heart failure may favor their direct physical interaction with the SERCA/PLN complex, affecting Ca 2+ regulation and cardiac contractility.
Irrespective of the possible role of the endogenous miRNAs toward the SERCA/PLN complex in cardiomyocytes, the nanomolar affinity of ssDNA for PLN and its ability to reverse its inhibitory effects on SERCA constitutes a unique opportunity to exploit oligonucleotides as scaffolds for the design of small molecules to target Ca 2+ regulation. While DNA aptamers or miRNA constructs have been successfully used to target and regulate the PLN gene 11,41 , silencing or ablation of the PLN gene has resulted in cardiomyopathies. In contrast, tunable regulation of PLN inhibitory function of SERCA by oligonucleotide-based drugs may represent a more promising therapeutic avenue in which the extent of SERCA activation can be fine-tuned to match the pathological insult.
Scientific RepoRts | 5:13000 | DOi: 10.1038/srep13000 Methods Wild type phospholamban expression and purification was achieved through the previously published methods 42 . Isotope labeling was accomplished via 13 C-enriched glucose and 15 N-enriched ammonium chloride. Calcium ATPase (SERCA1a isoform) was extracted from rabbit skeletal muscle according to previously reported procedure and purified by affinity chromatography using Reactive Red 43 . Pig cardiac SR vesicles (Lindenfelser's Meats, Monticello, MN, USA) were prepared as reported previously 44 . Lipids were obtained from Avanti Polar Lipids (Alabaster, AL). Single stranded DNA (ssDNA) sequences were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). Other chemicals were from Sigma Aldrich (St. Louis, MO).

SERCA Activity Assays.
Recombinant PLN was co-reconstituted with SERCA in multilamellar vesicles of 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleyl-sn-glycero-3-p hosphoethanolamine (DOPE) at 4:1 DOPC:DOPE molar ratio. A solution of ssDNA in deionized water was added directly to the vesicles and incubated for 20 minutes at 25 °C prior to starting the assay. The molar ratio of the components were 700:10:1:1 (lipid : PLN : SERCA : DNA). Calcium dependence of the SERCA ATPase activity was measured at 25 °C using a coupled enzyme assay 45 utilizing λ 340 absorbance (NADH) as an analytical signal using a Spectromax microplate reader (Molecular Devices). The initial rate was measured as a function of calcium concentration, and the data were fit using the Hill equation (1): where: V is the initial rate; V max is the maximum rate; pCa is the negative logarithm of calcium concentration; pK Ca is the pCa value where V = V max /2, and n is Hill coefficient. For comparison, each data set was normalized to V max after fitting. Inhibitory effects are expressed as changes in pK Ca relative to SERCA alone. Activity assays of SERCA2a were performed directly with pig cardiac SR, containing ~20 μ g of total protein (bicinchoninic acid assay). Cardiac SR vesicles were diluted to 100 μ l with buffer and incubated with 1 μ M of ssDNA (80mer) for 30 min prior to measurements. were prepared by co-mixing the proteins at a 1:1 molar ratio in 1% C 12 E 8 , and adding them to 10 mg of DMPC-d 54 solubilized with 1% C 12 E 8 . Next, ssDNA suspended in ddH2O was added and the detergent was removed by 3 hr incubation with BioBeads SM2 at a 30:1 Biobeads:C 12 E 8 ratio at room temperature. The sample was diluted to 50 mL with buffer, pelleted by centrifugation at 4 °C (100,000 × g, 30 minutes) and the pellet was packed in the MAS rotor. Isotropic bicelle samples for solution NMR were prepared by dissolving 1 mg of [U-13 C, 15  glycol monododecyl ether (C 12 E 8 ) at pH 7.0. Binding buffer additionally contained 0.25 mM DTT, 1 mM MgCl 2 , 1 mM KCl, and 5 mM CaCl 2 . Fluorescently labeled ssDNA samples in binding buffer (2.5 nM) were titrated with PLN and injected into a 50 cm × 50 μ m fused silica capillary (Polymicro Technologies, Phoenix, AZ) using hydrodynamic injection (1 psi for 4 s). Separations were performed at reversed polarity, 30 kV for 10 min. Electropherograms were analyzed using Cutter 7.0; peak heights of the free ssDNA peaks were used to calculate the bound fractions according to the equation (2)  FRET Experiments in reconstituted systems. SERCA was incubated with 1,5-I-AEDANS (Invitrogen), which specifically interacts with Cys 674 of the ATPase. The acceptor, Dabcyl-SE, was added at a 20-fold excess to WT PLN in 1% SDS, 100 mM NaHCO 3 , pH 9.0. The reaction was performed for 16 hours, and the labeled protein was purified using reverse phase HPLC. The steady-state fluorescence wavelengths were 350 nm (excitation) and 400-600 nm (emission).
FRET in-cell Binding Assay. N-termini of canine SERCA2a and canine PLN were fused with either mCerulean (Cer), or enhanced yellow fluorescent protein (YFP) 23 . Phospholamban oligomerization, quaternary structure, and SERCA binding were measured by fluorescence resonance energy transfer in living cells (line AAV-293). Cells were cultured in complete DMEM growth medium with 10% fetal bovine serum, 1% L-glutamine and incubated at 37 °C under 5% CO 2 . The cultured cells were subjected to transient transfection using the MBS mammalian transfection kit (Stratagene, La Jolla, CA). Cells were co-transfected with plasmids encoding Cer-PLN-AFA and YFP-PLN-AFA, or Cer-SERCA and YFP-PLN-WT with a molar ratio of 1:5 or 1:20 respectively 26 , either in the presence or absence of 1 μ M unlabeled 50-mer ssDNA. Following transfection, the cells were mildly trypsinized, resuspended in DMEM growth medium, plated on poly-D-lysine-coated glass bottom dishes, and allowed to adhere for 2 hours before imaging, as described previously 24 . The effect of ssDNA on PLN oligomerization and interaction with SERCA was quantified in live cells using wide-field fluorescence microscopy by acceptor sensitization FRET (EFRET) as described previously 24,52 . MetaMorph software was used to acquire a montage of 48 images using 40X objective having a numerical aperture of 0.75 and a motorized stage (Prior, Rockland, MA). Focus was automatically maintained by an optical feedback system (Perfect Focus System, Nikon). The exposure time was 150 ms for each channel: Cer, YFP, and FRET (Cer excitation/ YFP emission). FRET efficiency was calculated according to the equation (4) where: I DD and I AA are the fluorescence emission intensities of the donor channel (472/30 nm) with excitation of 427/10 nm, and the acceptor channel (542/27 nm) with excitation of 504/12 nm, respectively; I DA is the fluorescence emission intensity of the FRET channel (542/27 nm) with excitation of 427/10 nm. The constants a and d are cross-talk coefficients determined from acceptor-only or donor-only control samples respectively. G represents the ratio of the sensitized emission to the corresponding amount of donor recovery. We obtained values of 0.085, 0.737 and 4.6 for a, d, and G ratio respectively. The effect of ssDNA on parameters related to structure and binding affinity of PLN oligomer and SERCA-PLN regulatory complex were quantified by performing an 'in-cell' binding assay as described previously 24 . The FRET efficiency of individual cells co-expressing Cer-PLN-AFA and YFP-PLN-AFA, or Cer-SERCA and YFP-PLN-WT was plotted against relative protein concentration, quantified from the observed YFP fluorescence intensities. The cell-by-cell concentration dependence of FRET was fit to a hyperbolic curve to obtain the values of FRET max , K D 1, and K D 2. The maximal FRET (FRET max ) is the intrinsic FRET of the protein complex, providing an estimate of average distances between the binding partners. K D is the dissociation constant of the protein complex, providing an estimate of the apparent binding affinity. K D 1 is the apparent dissociation constant of the PLN-PLN oligomer and K D 2 is the apparent dissociation constant of the PLN-SERCA regulatory complex. For visualization, data from individual cells were pooled and the pooled data were fit by a hyperbolic or Hill function. Statistical Analysis. The data is pooled from 8 independent experiments for PLB-PLB FRET and 4 independent experiments for SERCA-PLB FRET using approximately 400-1200 cells per sample for each experiment. Errors are reported as standard error of the mean and statistical significance was evaluated using Student's T test, where p < 0.05 was considered significant.