Reliable Fluorometric Detection of SARS-CoV-2 by Targeting the G-Quadruplex through pH-Triggered Conformational Polymorphism

Abstract

Unravelling unique molecular targets specific to viruses is challenging yet critical for diagnosing emerging viral diseases. Nucleic acids and proteins are the major targets in diagnostic assays of viral pathogens. Identification of novel sequences and conformations of nucleic acids as targets is desirable for developing diagnostic assays specific to a virus of interest. Here, we disclose the identification and characterization of a highly conserved antiparallel G-quadruplex (GQ)-forming DNA sequence present within the SARS-CoV-2 genome. The two-quartet GQ with unique loop compositions formed a distinct recognition motif. Design, synthesis, and fine tuning of structure−activity of a set of small molecules led to the identification of a benzobisthiazole-based fluorogenic probe which unambiguously recognizes the target SARS-CoV-2 GQ DNA. A robust cost-effective assay was developed through thermal cycler PCR-based amplification of the antiparallel GQ-forming ORF1ab region of the SARS-CoV-2 genome and endpoint fluorescence detection with the probe. An exclusive pH window (3.5−4) helped trigger reliable conformational polymorphism (RCP) involving DNA duplex to GQ transformation, which aided the development of a GQ-RCP platform for the diagnosis of SARS-CoV-2 clinical samples. This general strategy can be adapted for the development of specific diagnostic assays targeting different noncanonical nucleic acid sequences.

G-Quadruplex Structures as a “Switch” Regulate ATF4 Expression in Ferroptotic HepG2 Cells

“…32 Doxorubicin, a clinically approved drug against SARS-CoV-2 infection, also was known as a G-quadruplex ligand, which provided potential clinically targets for the prevention and treatment of SARS-CoV-2. 33,34 In our study, the activity of ATF4 expression was enhanced in ferroptotic HepG2 cells induced by erastin. In the meantime, the preliminary bioinformatics analyses suggested that the regions of the ATF4 promoter also contained characteristic forming-motifs of G4.…”

“…Moreover, the functional G4 structures contained in the long terminal repeat (LTR) promoter regions could significantly inhibit the replication of HIV-1, and the BRACO-19 ligand exhibited striking anti-HIV-1 activity . Doxorubicin, a clinically approved drug against SARS-CoV-2 infection, also was known as a G-quadruplex ligand, which provided potential clinically targets for the prevention and treatment of SARS-CoV-2. , …”

Recent advances in applying G-quadruplex for SARS-CoV-2 targeting and diagnosis: A review

“…This sequence changes from the duplex form to the G4 structure in a pH-dependent manner. Thereafter, the authors developed a reliable fluorometric detection of SARS-CoV-2 by targeting this DNA G4 through pH-triggered conformational changes [157] . Furthermore, G4-based aptamers against proteins have been used in disease diagnosis and therapy [153] .…”

A Photoinduced Electron Transfer-Based Hypochlorite-Specific Fluorescent Probe for Selective Imaging of Proinflammatory M1 in a Rheumatoid Arthritis Model

“…However, differentiating macrophages (M1 and M2) is mainly based on different biomarkers like the cytokine expressed in the two different phenotypes of macrophages. − This involves immunohistochemistry, which is tedious and expensive. As an alternative, small molecular fluorescent probes − are promising diagnostic tools due to their simplicity, cost-effectiveness, and high sensitivity with good resolution. Moreover, a molecular fluorescent probe detecting hypochlorite (OCl – ) that can differentiate different macrophages is not known, as per our knowledge.…”