Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells.
Jin-Zhi ZhangNing-Ning ZhaoZi-Yue WangJuan HuShuangshuang ZhangPublished in: The Analyst (2023)
The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10 -6 U μL -1 . Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.
Keyphrases
- nucleic acid
- single molecule
- drug discovery
- transcription factor
- circulating tumor
- dna repair
- cell free
- living cells
- label free
- binding protein
- high intensity
- emergency department
- dna damage
- quantum dots
- small molecule
- soft tissue
- genome wide
- high throughput
- heavy metals
- sensitive detection
- circulating tumor cells
- single cell
- fluorescent probe