Isothermal cross-boosting extension–nicking reaction mediated exponential signal amplification for ultrasensitive detection of polynucleotide kinase. Issue 10 (28th April 2020)

• Record Type: Journal Article
• Authors: Li, Xiao-Yu; Cui, Yun-Xi; Du, Yi-Chen; Tang, An-Na; Kong, De-Ming
• Is Part Of: Analyst. Volume 145:Issue 10(2020)
• Page Start: 3742
• Page End: 3748
• Publication Date: 2020-04-28
• Subjects: Chemistry, Analytic -- Periodicals; 543
• DOI: 10.1039/c9an02569c
• Languages: English
• ISSNs: 0003-2654

Abstract

Abstract : 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. Abstract : 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.