Pre-Folded G-Quadruplex as a Tunable Reporter to Facilitate CRISPR/Cas12a-Based Visual Nucleic Acid Diagnosis

Tiantian Yang, Juan Li*, Decai Zhang, Xiaoxue Cheng, Jia Li, Xiaolin Huang*, Shijia Ding, Ben Zhong Tang*, Wei Cheng*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or "signal-on"colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection.

Original languageEnglish
Pages (from-to)3710-3719
Number of pages10
JournalACS Sensors
Volume7
Issue number12
DOIs
Publication statusPublished - 23 Dec 2022
Externally publishedYes

Keywords

  • CRISPR/Cas12a
  • G-quadruplex
  • label-free
  • signal-on
  • visual nucleic acid diagnosis

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