Yong-Joo Jeong

Isothermal DNA amplification in vitro: the helicase-dependent amplification system

Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification.

Isolation of inhibitory RNA aptamers against severe acute respiratory syndrome (SARS) coronavirus NTPase/Helicase

Abstract Recent outbreak of Severe Acute Respiratory Syndrome (SARS) that caused almost 800 victims requires a development of efficient inhibitor against SARS coronavirus (SCV). In this study, RNA aptamers against SCV NTPase/Helicase (nsP10) were isolated from RNA library containing random sequences of 40 nts using in vitro selection technique. Nucleotide sequences of enriched RNA aptamer pool (ES15 RNA) contain AG-rich conserved sequence of 10–11 nucleotides [AAAGGR(G)GAAG; R, purine base] and/or additional sequence of 5 nucleotides [GAAAG], which mainly reside at the loop region in all the predicted secondary structures. Isolated RNAs were observed to efficiently inhibit double-stranded DNA unwinding activity of the helicase by up to ∼85% with an IC50 value of 1.2nM but show a slight effect on ATPase activity of the protein in the presence of cofactor, poly (rU). These results suggest that the pool of selected aptamers might be potentially useful as anti-SCV agents.

Ursolic acid and its natural derivative corosolic acid suppress the proliferation of APC-mutated colon cancer cells through promotion of β-catenin degradation

Ursolic acid (UA) and corosolic acid (CA), naturally occurring pentacyclic triterpene acids, exhibit antiproliferative activities against various cancer cells, but a clear chemopreventive mechanism of these triterpenoids in colon cancer cells remains to be answered. Here we used a cell-based reporter system for detection of β-catenin response transcription (CRT) to identify UA as an antagonist of the Wnt/β-catenin pathway. UA promoted the degradation of intracellular β-catenin that was accompanied by its N-terminal phosphorylation at Ser33/37/Thr41 residues, marking it for proteasomal degradation. Consistently, UA down-regulated the intracellular β-catenin level in colon cancer cells with inactivating mutations of adenomatous polyposis coli (APC). In addition, UA repressed the expression of β-catenin/T-cell factor (TCF)-dependent genes, thereby inhibiting cell proliferation in colon cancer cells. The functional group analysis revealed that the major structural requirements for UA-mediated β-catenin degradation are a carboxyl group at position 17 and a methyl group at position 19. Notably, CA (2α-hydroxyursolic acid) was also found to decrease the level of intracellular β-catenin and to suppress the growth of APC-mutated colon cancer cells. Our findings suggest that UA and CA exert their anticancer activities against colon cancer cells by promoting the N-terminal phosphorylation and subsequent proteasomal degradation of β-catenin.

Identification of human UGT isoforms responsible for glucuronidation of efavirenz and its three hydroxy metabolites.

Uridine 5′-diphosphate-glucuronosyltransferases (UGTs) involved in the glucuronide formation of efavirenz (EFV) and its three hydroxy metabolites, 8-hydroxyefavirenz (8-OH EFV), 7-hydroxyefavirenz (7-OH EFV), and 8,14-dihydroxyefavirenz (8,14-diOH EFV), were assessed. Among 12 recombinant UGT isoforms tested, only UGT2B7 showed catalytic activity in the formation of EFV-N-glucuronide (EFV-G) as previously reported. On the other hand, almost all UGT isoforms were involved in the glucuronidation of the three hydroxy metabolites, although their relative contribution is unclear. The catalytic activities in the formation of EFV-G by 17 different human liver microsomes exhibit a more than 40-fold inter-individual variability, whereas those of glucuronidation of the three hydroxy metabolites showed almost identical activity. The formation of EFV-G showed a significant correlation (r = 0.920; p < 0.0001) with UGT2B7-catalysed azidothymidine glucuronidation in 17 different human liver microsomes. Furthermore, fluconazole, a known UGT2B7 inhibitor, potently inhibited the formation of EFV-G up to 80%. This suggests that EFV might be a specific UGT2B7 substrate in vitro. This is the first study identifying specific UGT isozymes that glucuronidate EFV and its three hydroxy metabolites. Continued identification and characterisation of these pathways may help reduce adverse effects such as CNS toxicity in EFV therapy.

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.

Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5′-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5′-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5′-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5′-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5′-overhang account for the observed enhanced processivity of DNA unwinding.

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.

Development of chemical inhibitors of the SARS coronavirus: viral helicase as a potential target.

Abstract Severe acute respiratory syndrome (SARS) was the first pandemic in the 21st century to claim more than 700 lives worldwide. However, effective anti-SARS vaccines or medications are currently unavailable despite being desperately needed to adequately prepare for a possible SARS outbreak. SARS is caused by a novel coronavirus, and one of its components, a viral helicase, is emerging as a promising target for the development of chemical SARS inhibitors. In the following review, we describe the characterization, family classification, and kinetic movement mechanisms of the SARS coronavirus (SCV) helicase—nsP13. We also discuss the recent progress in the identification of novel chemical inhibitors of nsP13 in the context of our recent discovery of the strong inhibition of the SARS helicase by natural flavonoids, myricetin and scutellarein. These compounds will serve as important resources for the future development of anti-SARS medications.

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.