aCt-oligonucleotide conjugation and enzyme loading

aCt was purchased from Sigma-Aldrich; NHS-C3-azide (BCL-014) and click reaction chemicals were a gift from baseclick GmbH. Enzyme-DNA conjugation was conducted in 2 steps: (i) enzyme conjugation with NHS-azide linker, and (ii) enzyme-azide CuAAC ‘click’ reaction with alkyne-modified oligonucleotide. First, aCt (14 lysine residues/molecule) was resuspended in aCt storage buffer (1 mM Tris/HCl, 2 mM CaCl₂, pH 8.0) at 100 µM final concentration; NHS-azide linkers were resuspended in DMSO at 10 mM final concentration. aCt (2 nmol) was incubated with NHS-C3-azide linker in lysine: linker = 1:10 molar ratios, and shaken (400 rpm) at 21 °C for 2 h in 50 µl aCt storage buffer final volume. The sample was then washed 2 times with aCt storage buffer by using Amicon Ultra-0.5 ml centrifugal filters (50 kDa) to remove unreacted NHS-C3-azide molecules. Next, 25 pmol aCt-azide conjugates were reacted with alkyne-modified DNA strand in lysine: DNA = 1:2 molar ratio—by using Oligo-Click-S-Basic reaction kit (baseclick GmbH)—and shaken (500 rpm) at 25 °C for 1 h in 12 µl final volume. Enzyme-DNA conjugates were analysed by non-reducing 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the DNA-conjugation yield. Enzyme concentration was calculated with NanoPhotometer P-300 spectrophotometer (Implen GmbH). 5 pmol purified alkyne-exposing DV were reacted with purified aCt-azide conjugates in 1:10 molar ratio by using Oligo-Click-S-Basic reaction kit (baseclick GmbH)—and shaken (500 rpm) at 25 °C for 1 h. The sample was split in 4 Oligo-Click-S-Basic reaction kit tubes for a final volume of 28.5 µl per tube. Excess enzyme was purified by using precipitation with PEG.