High-Fidelity and Simultaneous Sensing of Endogenous Mutant and Wild p53 Proteins for Precise Cancer Diagnosis and Drug Screening

Abstract

The simultaneous sensing of endogenous wild and mutant proteins plays a critical role in disease diagnosis and drug screening, and this remains a major current challenge. Here, we present a new and highly specific target-triggered dual proximity ligation assay (dPLA) strategy for sensitive and simultaneous sensing of wild and mutant p53 proteins from cancer cells. Two proximity DNA probes bind the target protein to form the primer/circular DNA template complexes with two nicks in the presence of the hairpin and ssDNA connector sequences via the strand displacement reaction. Only when the two nicks are simultaneously ligated can the rolling circle amplification be triggered with high fidelity for yielding substantially enhanced fluorescence. By encoding the hairpin sequence, two distinct fluorescence signals can be generated for simultaneous detection of the wild and mutant p53 proteins. Importantly, our method significantly reduces the possibility of nonspecific ligation reactions by using two ligation nicks, which minimizes the background noise. With this dPLA method, the regulation transition of intracellular mutant p53 to wild p53 proteins upon anticancer drug treatment has also been demonstrated, highlighting its usefulness for potential early disease diagnosis and drug screening with high fidelity.

Size-Discriminative DNA Nanocage Framework Enables Sensitive and High-Fidelity Imaging of Mature MicroRNA in Living Cells

“…For sensitive and robust miRNA detections, sensing strategies have been engineered to integrate with many amplification techniques, such as rolling circle amplification, enzyme-mediated amplification, and toehold-mediated strand displacement reaction (TSDR). − As a typical reaction driven by entropy, TSDR, in which a single strand binds with the short single-stranded region (called the toehold) of a double-stranded DNA to facilitate branch migration, has attracted tremendous attention since the amplification reaction occurs under enzyme-free and isothermal conditions, making the technique particularly adaptable to live-cell imaging. , For instance, TSDR amplification for intracellular RNA imaging driven by the fuel DNA strands that were transfected into live cells with the assistance of liposome 3000 has been demonstrated. , Although these cascaded amplification processes have made significant advances in biological sensing applications, they necessitate the additional delivery of exogenous fuel strands, complicating the sensing designs and operational procedures. Noteworthily, adenosine triphosphate (ATP), which is abundant (1–10 mM), essential, and an endogenous biomolecule in cells, is ideally suitable as fuel molecules for driving the cascaded TSDR in living cells. − The challenge for accurate and reliable detection of mature miRNAs is that the detection process is usually accompanied by the degradation of sensing probes or false-positive signals from the interferences of pre-miRNA molecules that contain the sequences of the mature miRNAs. − Alternatively, inspired by the size differences between mature miRNAs (18–25 nt) and pre-miRNAs (60–70 nt), the size-discriminative sensing strategies on the basis of the size differences between nontarget molecules and target molecules are promising .…”

New insight into the application of fluorescence platforms in tumor diagnosis: From chemical basis to clinical application

“…However, the potentially high background noise of RCA remains a significant challenge. Fang Yang's group 140 This is of great significance to the treatment of tumors and the development of antitumor drugs.…”

“…(ii) Fluorescence spectra of miRNA‐155 and miRNA‐150 detected by QD‐MB@MoS 2 probe in different concentration buffers, and the relationship between the fluorescence ratio and the logarithm of the double miRNA concentration (F0155 and F0150 represent the fluorescence intensity of two miRNA detection probes respectively; F155 and F150 represent the fluorescence intensity of miRNA‐155 and miRNA‐150 simultaneously measured). (E) p53 protein detection platform 140 (i) The detection mechanism of the probe for wild‐type p53 protein and mutant p53 protein; (ii) Fluorescence signals of MDA‐MB‐231 cell lysates after 48 h of treatment with 100 μM DOX and different concentrations of PRIMA‐1 drug. BSA, Bovine albumin; CEA, carcinoembryonic antigen; CTC, circulating tumor cell; DOX, doxorubicin; IgG, Immunoglobulin G; miRNA, microRNA; MOF, metal‐organic framework; QD, quantum dot; ssDNA, single－stranded DNA.…”

“…However, the potentially high background noise of RCA remains a significant challenge. Fang Yang's group 140 adopted a highly specific dual PLA (dPLA) strategy triggered by proximity‐binding‐induced strand displacement reaction (PBI‐SDR) to achieve high‐sensitivity detection of endogenous Mp53 and Wp53 proteins simultaneously. It has great application potential in early tumor screening and evaluation of antitumor drug efficacy.…”

Swelling of Serum-Stable DNA Nanoparticles upon Target-Induced Conformational Rearrangement of Sensing Probes for the Signal-On Detection of Cancer-Related Genes

“…The detection of cancer-related nucleic acids is fundamental for studying their biological functions and exploiting molecular diagnostics . For example, the p53 gene is a well-known tumor suppressor gene and plays a significant role in DNA repair, cell differentiation, cell cycle, apoptosis, and other biological functions by coding and expressing the p53 protein. − Meanwhile, the detection of nucleic acids has been found to have a wide range of applications, including early diagnosis of cancers, drug discovery, pathological research, forensic science, and environmental monitoring . Therefore, there has been tremendous interest in developing sensitive, selective, simple, and cost-effective biosensors for the detection of nucleic acids.…”