Jin-Moo Lee

Single-stranded DNA aptamer that specifically binds to the influenza virus NS1 protein suppresses interferon antagonism.

Non-structural protein 1 (NS1) of the influenza A virus (IAV) inhibits the hosts innate immune response by suppressing the induction of interferons (IFNs). Therefore, blocking NS1 activity can be a potential strategy in the development of antiviral agents against IAV infection. In the present study, we selected a single-stranded DNA aptamer specific to the IAV NS1 protein after 15 cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure and examined the ability of the selected aptamer to inhibit the function of NS1. The selected aptamer binds to NS1 with a Kd of 18.91±3.95nM and RNA binding domain of NS1 is determined to be critical for the aptamer binding. The aptamer has a G-rich sequence in the random sequence region and forms a G-quadruplex structure. The localization of the aptamer bound to NS1 in cells was determined by confocal images, and flow cytometry analysis further demonstrated that the selected aptamer binds specifically to NS1. In addition, luciferase reporter gene assay, quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that the selected aptamer had the ability to induce IFN-β by suppressing the function of NS1. Importantly, we also found that the selected aptamer was able to inhibit the viral replication without affecting cell viability. These results indicate that the selected ssDNA aptamer has strong potential to be further developed as a therapeutic agent against IAV.

Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13.

Abstract Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors.

Aryl diketoacids (ADK) selectively inhibit duplex DNA-unwinding activity of SARS coronavirus NTPase/helicase.

Abstract As anti-HCV aryl diketoacids (ADK) are good metal chelators, we anticipated that ADKs might serve as potential inhibitors of SARS CoV (SCV) NTPase/helicase (Hel) by mimicking the binding modes of the bismuth complexes which effectively competes for the Zn2+ ion binding sites in SCV Hel thereby disrupting and inhibiting both the NTPase and helicase activities. Phosphate release assay and FRET-based assay of the ADK analogues showed that the ADKs selectively inhibit the duplex DNA-unwinding activity without significant impact on the helicase ATPase activity. Also, antiviral activities of the ADKs were shown dependent upon the substituent. Taken together, these results suggest that there might be ADK-specific binding site in the SCV Hel, which warrants further investigations with diverse ADKs to provide valuable insights into rational design of specific SCV Hel inhibitors.

Complementary Silicon Nanowire Hydrogen Ion Sensor With High Sensitivity and Voltage Output

Complementary Si nanowire (SiNW) hydrogen ion sensors with high sensitivity and robust voltage output are demonstrated on a 6-in silicon-on-insulator wafer using a conventional wafer-level top-down process. The proposed SiNW sensors exhibit a logic threshold voltage shift of 88.9 mV/pH and an output voltage swing of 162 mV/pH. Furthermore, a simplified analytical model confirms that the proposed sensors have an output voltage swing that is 1.6 times larger than single SiNW sensor counterparts with a resistive load. Therefore, the proposed fabrication approach is expected to be a good solution for a very sensitive voltage readout scheme for the mass production of top-down processed biosensors.

Investigation of the pharmacophore space of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) NTPase/helicase by dihydroxychromone derivatives.

Abstract Aryl diketoacids have been identified as the first SARS-CoV NTPase/helicase inhibitors with a distinct pharmacophore featuring an arylmethyl group attached to a diketoacid. In order to search for the pharmacophore space around the diketoacid core, three classes of dihydroxychromone derivatives were prepared. Based on SAR study, an extended feature of the pharmacophore model of SARS-CoV NTPase/helicase was proposed which is constituted of a diketoacid core, a hydrophobic arylmethyl substituent, and a free catechol unit.

SiNW-CMOS Hybrid Common-Source Amplifier as a Voltage-Readout Hydrogen Ion Sensor

We proposed a silicon nanowire (SiNW)-CMOS hybrid common-source amplifier (CSA) as a voltage-readout hydrogen ion sensor and fabricated it on a 6-in silicon-on-insulator wafer using a conventional back-end process and a wafer-level top-down process. The current-defined sensitivity ΔIDS/IDS of the SiNW itself was 0.18, and the maximum output voltage shift found in the CSA configuration was 38.1 mV/pH. A simple analytical model showed that the voltage-defined sensitivity can be controlled and maximized by the gate voltage of the MOSFET, indicating that process variations of SiNWs are electrically controllable in a SiNW-based sensor array system with the voltage-readout scheme.

Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.

Characterization and Capacitive Modeling of Target Concentration-Dependent Subthreshold Swing in Silicon Nanoribbon Biosensors

The effect of streptavidin concentration as a target molecule on the subthreshold swing (SS) of biotin-functionalized silicon nanoribbon sensors was investigated for a configuration of field effect transistors. The increasing concentration of conjugated target molecules was modeled as a degradation of the coupling capacitance between the electrolyte and the sensor surface. A binding factor for the ratio of conjugation between targets and receptors was modeled as a coupled capacitance and experimentally determined to be from 0.65 (streptavidin at 1 pM) to 1 (1 nM). This result suggests that SS is potentially a good indicator of the target concentration.

Identification of a Novel Small Molecule Inhibitor Against SARS Coronavirus Helicase.

A new chemical inhibitor against severe acute respiratory syndrome (SARS) coronavirus helicase, 7-ethyl-8-mercapto-3-methyl-3,7-dihydro-1H-purine-2,6-dione, was identified. We investigated the inhibitory effect of the compound by conducting colorimetry-based ATP hydrolysis assay and fluorescence resonance energy transfer-based double-stranded DNA unwinding assay. The compound suppressed both ATP hydrolysis and double-stranded DNA unwinding activities of helicase with IC50 values of 8.66 ± 0.26 μM and 41.6 ± 2.3 μM, respectively. Moreover, we observed that the compound did not show cytotoxicity up to 80 µMconcentration. Our results suggest that the compound might serve as a SARS coronavirus inhibitor.

A Novel Chemical Compound for Inhibition of SARS Coronavirus Helicase

We have discovered a novel chemical compound, (E)-3-(furan-2-yl)-N-(4-sulfamoylphenyl) acrylamide, that suppresses the enzymatic activities of SARS coronavirus helicase. To determine the inhibitory effect, ATP hydrolysis and double-stranded DNA unwinding assays were performed in the presence of various concentrations of the compound. Through these assays, we obtained IC50 values of 2.09 ± 0.30 µM (ATP hydrolysis) and 13.2 ± 0.9 µM (DNA unwinding), respectively. Moreover, we found that the compound did not have any significant cytotoxicity when 40 µM of it was used. Our results showed that the compound might be useful to be developed as an inhibitor against SARS coronavirus.