Label-free ultrasensitive determination of EcoRI activity based on terminal deoxynucleotidyl transferase generated G-quadruplexes

In this study, a label-free ultrasensitive fluorescent biosensor for monitoring of EcoRI activity has been developed based on terminal deoxynucleotidyl transferase (TdT) directed G-quadruplexes formation. Double-stranded DNA is specifically cleaved by EcoRI into single-stranded fragments with the newly generated free 3′-OH terminus. In the condition of the polymerization pool containing 10% dTTP, 40% dATP and 50% dGTP, TdT can elongate the cleaved fragments at their 3′-OH ends to produce G-rich DNA sequences which could subsequently form into G-quadruplexes motif in the aid of K+. The obtained G-quadruplexes structure can be recognized by the typical G-quadruplex-selective probe N-methylporphyrin dipropionate IX (NMM). Thus, EcoRI activity could be easily and sensitively determined by using this detection platform. The detection limit for EcoRI is as low as 0.07 U/mL according to the linear concentration range of 0.1-30 U/mL. Moreover, the proposed assay showed high potential in real sample detection. This detection platform is label-free, highly sensitive, simple to operate and cost effective, possessing high application potential as a useful tool for endonuclease detection. Moreover, we also demonstrated the capability of this strategy to detect EcoRI in serum sample, showing high application potential as a useful tool for endonuclease detection.