Fig. 3: Real-time monitoring of PARP1-dependent PARylation at various NAD⁺ concentrations.

a Chemical structure and respective IR spectra of NAD⁺, NMN and ADP-ribose. The band assigned to the nicotinamide moiety (1695 cm⁻¹) is indicated. b Representative time-dependent spectra following the addition of various NAD⁺ concentrations to PARP1 bound to immobilised DNA_blunt. Amide I (1645 cm⁻¹) and amide II (1548 cm⁻¹) bands of PARP1 and anti-symmetric (1236 cm⁻¹) and symmetric (1074 cm⁻¹) phosphate vibrations of generated PAR are indicated. c, d Evaluation of b. ‘0 min’ refers to start of measurements. c Dissociation of PARP1 from DNA_blunt upon addition of various NAD⁺ concentrations. Time-dependent intensity decrease of the amide II band (1548 cm⁻¹) was analysed. Intensity of the amide II band before addition of NAD⁺ was set to 100%. PARP1 dissociation parameters were calculated via a mono-exponential fit function. d PAR formation upon addition of various NAD⁺ concentrations. Time-dependent intensity increase of the anti-symmetric phosphate vibration of PAR (1236 cm⁻¹) was analysed. Data was normalised to the intensity of the amide II bands (1548 cm⁻¹) of PARP1 before addition of NAD⁺. PAR formation parameters were calculated via a mono-exponential fit function. c, d Data from same experiments. Means ± SEM of n = 3 (100 µM) and n = 2 (0, 1, 10 and 500 µM) independent experiments, respectively. Source data are provided as a Source Data file.