Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent "On-Off" switching DNA aptamer

Abstract

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 μM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.

Keywords: ATP aptamer; ATP synthase inhibition; Adenosine triphosphate; Intramolecular fluorescence resonance energy transfer (iFRET); Microscopic images; Oligomycin.

Graphical abstract

Fig. 1

a Principle of ATP aptamer operation. b Fluorescence spectra for ATP aptamer after addition of ATP at different concentrations, C_ATP (mM): (1) 0, (2) 0.033, (3) 0.066, (4) 0.1, (5) 0.13, (6) 0.16, (7) 0.2, (8) 0.23, (9) 0.26, (10) 0.3, (11) 0.33. c Dependence of I_FL vs. C_ATP. d Stern-Volmer plot. e Dependence of I_FL vs. C_ATP for C_Apt = 16.7 nM. f Dependence of log (I_FL,0 - I_FL)/I_FL vs. log C_ATP; conditions: C_Apt = 66.7 nM; 20 mM Tris-HCl with 100 mM NaCl and 5 mM MgCl₂, pH 7.4; λ_ex = 480 nm, λ_em = 516 nm

Fig. 2

a Four secondary structures of Apt(ATP) obtained in 100 mM NaCl with 5 mM MgCl₂ solution at 25 °C using UNAFold program. b Tertiary structure of Apt(ATP)

Fig. 3

a Schematic view of the formation of Apt(ATP)@LIP for ATP recognition and ATP detection in cells. b–g Detection of ATP content in fixed SW480 cells untreated and treated with 1 and 5 μM concentration of oligomycin (OMC) for 20 h and transfected with Apt(ATP)@LIP for 135 min, b–d light-dark field, e–g dark field. h Morphology of the untreated SW480 cells. i Dark field fluorescence image of untreated SW480 cells (control). j Comparison of the relative intensity of fluorescence for SW480 cells transfected with Apt(ATP)@LIP after addition of OMC, C_OMC (μM) (1) 0, (2) 1.0, (3) 5.0, with incubation time t_inc = 20 h

Fig. 4

a Schematic illustration of mechanism of oligomycin (OMC) action in mitochondrial respiratory chain site. b Effect of OMC and FCCP on mitochondrial oxygen consumption reflecting ATP synthase activity in SW 480 cell line. c Structure of OMC. d Structure of FCCP. Conditions: C_OMC = 0.3 and 1 μM; C_FCCP = 1 μM, n = 3. ***Significantly different to baseline values with p < 0.01. NS—1 μM OMC values not significant to oligomycin 0.3 μM values

Fig. 5

a Dependence of normalized fluorescence intensity of an ATP aptamer on concentration of nucleoside triphosphates: (1) ATP, (2) CTP, (3) UTP, (4) GTP. b Block diagram of selectivity coefficient for analytes C_Analytes = 330 μM. Conditions: C_Apt = 66.7 nM, 20 mM Tris-HCl buffer with 100 mM NaCl and 5 mM MgCl₂, pH 7.4

Fig. 6

The results of MTT cell viability tests for cells treated with 0.3 μM, 1 μM, and 5 μM OMC; positive control: (OMC) = 0 μM; negative control: (H₂O₂) = 8 M

Fig. 7

Test of Apt(ATP) sensitivity to the addition of ATP to a lysate solution of SW480 cells