Detection of Small-Sized DNA Fragment in a Glassy Nanopore by Utilization of CRISPR-Cas12a as Converter System

Abstract

The fabrication of nanopore with matched pore size, and the existence of multi interferents make the reproducible detection of small-sized molecules by means of solid-state nanopore still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructure related to the existence of small-sized target. In particular, DNA tetrahedron with well-defined 3D nanostructure is the ideal candidate to be used as signal transducer. Here, we demonstrate the detection of L1-encoding gene of HPV18 as test DNA target sequence in a reaction buffering solution, where long single-stranded DNA linking DNA tetrahedrons onto the surface of magnetic beads are cleaved by target DNA-activated CRISPR-cas12 system. The DNA tetrahedrons are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) One signal transducer can be used to detect different targets; (2) Glassy nanopore with pore size much larger than the target DNA fragment can boost the tolerance of contaminants and interferents which often degrade the performance of nanopore sensor.