Multifunctional nanocomposite system with positing and Mn2+-enhanced-Cas12a shearing for precise and efficient intracellular imaging

Abstract

The CRISPR/Cas12a fluorescence system is commonly used for in vitro detection, and its application in cells is challenging due to the difficulty in achieving precise localization and good reaction kinetics. This work creatively developed a multifunctional nanocomposite system (MNCS) with positing and Mn²⁺-enhanced-Cas12a shearing for precise and efficient intracellular imaging of microRNA-155 (miRNA-155). The rolling circle amplification (RCA) was well-engineered for generating a long single strand DNA (ssDNA), which was encoded with abundantly repetitive AS1411 aptamers for targeting cancer cells and functional sequences for activating Cas12a. This long ssDNA coupled MnO₂ nanoparticles (NPs), FAM and BHQ1 labeled reporter probes and CRISPR RNA/Cas12a (crRNA/Cas12a) to construct MNCS. AS1411 aptamers on ssDNA improved the targeting ability of MNCS, thereby reducing interference caused by mistargeted cells. As MNCS entered target cells, intracellular glutathione (GSH) converted MnO₂ into Mn²⁺, causing the disintegration of MNCS. In this case, the “lock-in state” crRNA hybridized with intracellular miRNA-155 to expose the binding site on crRNA so that it bound with functional sequences on ssDNA. Under the accelerated action of Mn²⁺, the trans-cleavage activity of Cas12a was highly activated for shearing reporter probes, thus recovering fluorescence signal of FAM. The MNCS with positing and Mn²⁺-enhanced-Cas12a shearing allowed in vitro detection of miRNA-155 with a low limit of detection of 4.67 pM, and also enabled highly sensitive intracellular imaging of miRNA-155. The coupling of targeting capability of AS1411 aptamers with significantly enhanced reaction kinetics of CRISPR/Cas12a system by Mn²⁺ provides a promising intracellular imaging strategy for nucleic acid.