Self-activation of symbiotic multi-DNA machines transducing exponentially amplified fluorescence for ultrasensitively signaling of terminal deoxynucleotidyl transferase activity

DNA machines with great design flexibility and precise programmability have exhibited promising potential for biomedical analysis. In this contribution, we engineered a symbiotic multi-DNA machine (SM-DNA machine) for ultrasensitively analysis of terminal deoxynucleotidyl transferase (TdT) activity. The machine, made from only one poly-thymine (Poly-T) tailed functional hairpin probe (T-HP) to simplify the machine composition, is dependent on the template-free elongation property of TdT. This design ensures that in the “off” state there are no physical links between intermolecular T-HPs. Introduction of external TdT would elongate the T-HP with a long poly-adenine chain to trigger the intermolecular interactions between different T-HPs, which tuned the SM-DNA machine to an “ON” state responsible for the creation of multiple cyclic signal amplification processes of strand replication, cleavage, and displacement. Such multiple machine-like movements result in an exponentially amplified fluorescence signal to ensure the system has a limit of detection as low as 0.01 U/mL. The TdT based strand elongation ensures the machine has an excellent specificity to discriminate TdT from other enzymes. We expect this DNA machine with an excellent assay performance for TdT biomarker biosensing could be readily expanded as a universal platform for various sensing applications and disease diagnosis.