Thiolate DNAzymes on Gold Nanoparticles for Isothermal Amplification and Detection of Mesothelioma-derived Exosomal PD-L1 mRNA

Abstract

Catalytic DNAzymes have been used for isothermal amplification and rapid detection of nucleic acids, holding the potential for point-of-care testing applications. However, when Subzymes (universal substrate and DNAzyme) are tethered to the polystyrene magnetic microparticles via biotin−streptavidin bonds, the residual free Subzymes are often detached from the microparticle surface, which causes a significant degree of false positives. Here, we attached dithiol-modified Subzyme to gold nanoparticle and improved the limit of detection (LoD) by 200 times compared to that using magnetic microparticles. As a proof of concept, we applied our new method for the detection of exosomal programed cell-death ligand 1 (PD-L1) RNA. As the classical immune checkpoint, molecule PD-L1, found in small extracellular vesicles (sEVs, traditionally called exosomes), can reflect the antitumor immune response for predicting immunotherapy response. We achieved the LoD as low as 50 fM in detecting both the RNA homologous to the PD-L1 gene and exosomal PD-L1 RNAs extracted from epithelioid and nonepithelioid subtypes of mesothelioma cell lines, which only takes 8 min of reaction time. As the first application of isothermal DNAzymes for detecting exosomal PD-L1 RNA, this work suggests new point-of-care testing potentials toward clinical translations.

Modular DNAzymes-Hydrogel Membrane Carriers for Highly Sensitive Isothermal Cross-Cascade Detection of Pathogenic Bacteria Nucleic Acids

“…8,13 Our team recently has been successful in conjugating thiolate DNAzymes to gold nanoparticles. 6 By leveraging self-deactivation properties of DNAzymes and initiating multicomponent DNAzymes, we have achieved the detection of PD-L1 RNA target sequence with sensitivity down to 50 fM. The mechanisms on the DNAzyme deactivation on magnetic particles or gold nanoparticles remain unclear, being arguably ascribed to the high DNAzyme attachment density and steric hindrance.…”

“…DNAzymes carriers not only are essential for enzyme immobilization, isolation, and transport but also facilitate the design of highly sensitive assay strategies with signal propagation. To date, DNAzyme carriers such as gold , and magnetic particles − have been reported. The detection mechanism for nucleic acid targets has been majorly based on peroxidase-like DNAzymes attached to the particle carriers to generate chemiluminescence via the oxidation of lumino. , Our team recently has been successful in conjugating thiolate DNAzymes to gold nanoparticles .…”

“…To date, DNAzyme carriers such as gold , and magnetic particles − have been reported. The detection mechanism for nucleic acid targets has been majorly based on peroxidase-like DNAzymes attached to the particle carriers to generate chemiluminescence via the oxidation of lumino. , Our team recently has been successful in conjugating thiolate DNAzymes to gold nanoparticles . By leveraging self-deactivation properties of DNAzymes and initiating multicomponent DNAzymes, we have achieved the detection of PD-L1 RNA target sequence with sensitivity down to 50 fM.…”

Accurate Cancer Screening and Prediction of PD-L1-Guided Immunotherapy Efficacy Using Quantum Dot Nanosphere Self-Assembly and Machine Learning

“…Nucleic acid amplification techniques, such as multivalence-actuated DNA nanomachines and dual-aptamer activated proximity-induced droplet digital PCR, enable highly sensitive detection of PD-L1 by nucleic acid amplification triggered by aptamer recognition. Gene sequencing techniques, such as real-time quantitative fluorescence polymerase chain reaction (qPCR) and isothermal amplification, are used to detect PD-L1 gene expression levels to indirectly reflect the expression of the PD-L1 protein. Despite the great success of the above studies in quantitative PD-L1 detection, , these methods often require complex experimental steps and expensive equipment, and reliable methods for cancer screening and quantitative analysis of PD-L1 expression levels by circulating exosomes are still less reported.…”

A cubic DNA nanocage probe for in situ analysis of miRNA-10b in tumor-derived extracellular vesicles

“…5,6 In particular, tumor-derived EVs carry abundant genetic information from parental cells, such as diverse nucleic acids, proteins, and other bioactive molecules, which are closely related to tumor development and metastasis and can provide valuable information for early diagnosis of cancer, analysis of malignancy, tumor metastasis, and evaluation of therapeutic efficacy. [7][8][9][10] Recently, tumor-derived EV-encapsulated non-coding RNAs (such as miR-NAs) have become promising biomarkers for cancer diagnosis because they contribute to different stages of cancer progression and are well protected from degradation by RNases by the lipid bilayer of EVs. [11][12][13] EV-encapsulated miRNAs can be absorbed by adjacent or distant receptor cells, triggering changes in gene expression.…”