posted on 2022-01-27, 20:14authored bySumon Pratihar, Ragini Agrawal, Virender Kumar Pal, Amit Singh, Thimmaiah Govindaraju
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.