ac5b00810_si_001.pdf (2.35 MB)
Recognition of Dual Targets by a Molecular Beacon-Based Sensor: Subtyping of Influenza A Virus
journal contribution
posted on 2015-05-19, 00:00 authored by Chun-Ching Lee, Yu-Chieh Liao, Yu-Hsuan Lai, Min-Chieh ChuangA molecular
beacon (MB)-based sensor to offer a decisive answer
in combination with information originated from dual-target inputs
is designed. The system harnesses an assistant strand and thermodynamically
favored designation of unpaired nucleotides (UNs) to process the binary
targets in “AND-gate” format and report fluorescence
in “off–on” mechanism via a formation of a DNA
four-way junction (4WJ). By manipulating composition of the UNs, the
dynamic fluorescence difference between the binary targets-coexisting
circumstance and any other scenario was maximized. Characteristic
equilibrium constant (K), change of entropy (ΔS), and association rate constant (k) between
the association (“on”) and dissociation (“off”)
states of the 4WJ were evaluated to understand unfolding behavior
of MB in connection to its sensing capability. Favorable MB and UNs
were furthermore designed toward analysis of genuine genetic sequences
of hemagglutinin (HA) and neuraminidase (NA) in an influenza A H5N2
isolate. The MB-based sensor was demonstrated to yield a linear calibration
range from 1.2 to 240 nM and detection limit of 120 pM. Furthermore,
high-fidelity subtyping of influenza virus was implemented in a sample
of unpurified amplicons. The strategy opens an alternative avenue
of MB-based sensors for dual targets toward applications in clinical
diagnosis.