Isothermal cross-boosting extension-nicking reaction mediated exponential signal amplification for ultrasensitive detection of polynucleotide kinase

• Published in: Analyst (London) Vol. 145; no. 1; pp. 3742 - 3748
• Main Authors: Li, Xiao-Yu, Cui, Yun-Xi, Du, Yi-Chen, Tang, An-Na, Kong, De-Ming
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 21.05.2020
• Subjects: Adenine; Amplification; Binding sites; Biosensing Techniques - methods; DNA Probes - genetics; DNA Probes - metabolism; Elongation; Enlargement; Fluorescence; HeLa Cells; Humans; Kinases; Limit of Detection; Nucleic Acid Amplification Techniques; Oligonucleotides; Polynucleotide 5'-Hydroxyl-Kinase - metabolism; Substrates; Uracil
• ISSN: 0003-2654; 1364-5528; 1364-5528
• DOI: 10.1039/c9an02569c

A novel nucleic acid-based isothermal signal amplification strategy, named cross-boosting extension-nicking reaction (CBENR) is developed and successfully used for rapid and ultrasensitive detection of polynucleotide kinase (PNK) activity. Only two simple oligonucleotides (recognition substrate ( RS ) and TaqMan probe) are applied to construct the PNK-sensing platform. In the presence of PNK, the 3′-phosphate end of RS will be converted to the 3′-hydroxyl one, and then extended to a long poly-adenine (poly-A) sequence under the catalysis of terminal deoxynucleotidyl transferase (TdT). The poly-A sequence provides multiple binding sites for the TaqMan probe to form multiple DNA duplexes. Subsequently, ribonuclease HII (RNase HII) cuts the TaqMan probe into two parts at the pre-set uracil site, generating a fluorescence signal and providing new substrates for TdT elongation. The TdT-catalyzed substrate extension and RNase HII-catalyzed probe nicking are boosted by each other, resulting in persistent enlargement of these two reactions and thus giving ultrahigh signal amplification efficiency. Utilizing the CBENR-based PNK sensor, ultrasensitive detection of PNK activity was achieved with a detection limit as low as 3.0 × 10 −6 U mL −1 . Quantification of endogenous PNK activity at the single-cell level and the screening/evaluation of PNK inhibitors were also achieved. A novel nucleic acid-based isothermal signal amplification strategy, named cross-boosting extension-nicking reaction (CBENR) is developed and successfully used for rapid and ultrasensitive detection of polynucleotide kinase (PNK) activity.