1. Sensitive Autocatalytic Hybridization Circuit for Reliable In Situ Intracellular Polynucleotide Kinase Imaging
- Author
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Yanping Zhang, Ruomeng Li, Shanshan Yu, Jinhua Shang, Yuqiu He, Yushi Wang, Xiaoqing Liu, and Fuan Wang
- Subjects
Polynucleotide 5'-Hydroxyl-Kinase ,Bacteriophage T4 ,Nucleic Acid Hybridization ,Reproducibility of Results ,Biosensing Techniques ,DNA ,Analytical Chemistry - Abstract
Exploring the characteristic functions of polynucleotide kinase (PNK) could substantially promote the elucidation of PNK-related mechanistic pathways. Yet, the sensitive and reliable detection of intracellular PNK still presents a challenging goal. Herein, we propose a simple autocatalytic hybridization circuit (AHC) foriin situ/iintracellular imaging of PNK with high reliability. The AHC amplifier consists of two mutually activated hybridization chain reaction (HCR) modules for magnified signal transduction. The PNK is transduced into initiatorbI/bby phosphorylation and cleavage of mediatorbH/bsubbp/b/sub. InitiatorbI/bactivates the initial HCR-1 module, leading to the formation of long dsDNA nanowires that carry numerous initiatorbT/b. Then,bT/b-initiated feedback HCR-2 module generates branched products that contain plentiful initiatorbI/b, thus realizing an autocatalytic HCR amplification reaction. Simultaneously, the HCR-2 module is also assembled as a versatile signal transduction unit for generating the amplified readout. Based on the mutually sustained accumulation of two initiators for the reciprocal activation of two reaction modules, continuous signal amplification and assembly of high-molecular-weight copolymers endow the AHC system with high sensitivity and robustness for the PNK assay. Moreover, the PNK-sensing AHC system achieves reliable imaging of intracellular PNK, thus showing great potential to decipher the correlation between PNK and related diseases.
- Published
- 2022