title: Recognition of dual targets by a molecular beacon-based sensor: subtyping of influenza A virus.
abstract: A 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.
authors: †Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
authors: Chun-Ching
authors: CC
authors: Lee
authors: ‡Institute of Population Health Science, National Health Research Institutes, Zhunan, Miaoli County 35053, Taiwan.
authors: Yu-Chieh
authors: YC
authors: Liao
authors: †Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
authors: Yu-Hsuan
authors: YH
authors: Lai
authors: Chang-Chun David
authors: CC
authors: Lee
authors: †Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
authors: Min-Chieh
authors: MC
authors: Chuang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D053125:Influenza A Virus, H5N2 Subtype / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism; D057230:Limit of Detection; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D015345:Oligonucleotide Probes