The incidence of dengue has grown dramatically worldwide in recent decades,[1] making dengue fever a significant emerging public health problem for which no effective treatment exists. RNA helicase is responsible for almost all RNA metabolism, and helicase plays an essential role in virus replication. We propose to analyze the helicase sequence and obtain the NS3 protease with high activity by molecular cloning and protein purification. Finally, we aim to construct a high-throughput screening method in vitro that can be applied to the rapid discovery of helicase inhibitors.
We obtained the target fragment of NS3-HEL/NS3-ZIKA by PCR and the recombinant plasmid by enzyme ligation. The recombinant plasmids, pET-28a-NS3-Hel and pET-28a-NS3-ZIKA, were then transferred to BL21 for inducible gene expression, and the resulting protein was verified for function, as shown in Figure 1.
We designed two genes, NS3-Hel and NS3-ZIKA, and obtained a plasmid pET28a from the laboratory. We then amplified NS3-Hel and NS3-ZIKA genes with pET28a and constructed the plasmids by instrumentation. The target DNA was obtained through PCR. Then, enzyme digestion was performed, and the plasmid was converted into BL21(DE3) by heat shock. Finally, we transferred the plasmid into the BL21(DE3). As shown in Figure 2, NS3-Hel (1344bp) & NS3-ZIKA (1854bp), we obtained the specific plasmids we wanted. We verified that we got the correct gene by confirming the bp length as well as the sequencing results. We can use this as a precondition for further operations.To ensure that the plasmid construction is 100% correct, we sequenced the target genes that NS3-Hel and NS3-ZIKA.
We validated the recombinant plasmid by inserting DNA fragments into a plasmid (pET-28a), as shown in Figure 3a. The data confirms that the fragments are in the correct position. After transferring the recombinant plasmid into BL21 to induce its expression by IPTG, we purified the two proteins to obtain two pure proteins by His-Tag.
As shown in Figure 3B, the size of the two recombinant proteins NS3-Hel / NS3-ZIKA is round 49 kDa as expected. Therefore, we can see that the proteins were successfully induced to express.
The Fluorescence Resonance Energy Transfer (FRET) is based on the resonance energy transfer of fluorescent proteins to study the structure and dynamics of proteins and nucleic acids, in the detection and visualization of intermolecular association and in the development of intermolecular binding assays.[2] In this project, the FRET method was used to test the activity of NS3 protein, where an FRET pair consisting of a donor and an acceptor fluorophore is attached to the ends of a double-stranded DNA or RNA substrate. When the helicase, NS3 protein unwinds the substrate, the donor and acceptor fluorophores move apart which will result in their distance reaching a critical range (typically 1-10 nanometers), and then FRET (a measurable decrease in fluorescence) occurs.
After purifying the protein, we reacted with ATP, MOPS Buffer, MgCl2, and double-chain substrate for 15 mins. We then measured the fluorescence. As the concentration of our protein increased, the fluorescence gradually decreased. As shown in Figure 4, our fluorescence tended to decline as our protein gradually increased since the NS3 protein . Therefore, we can prove that the NS3 protein possesses helicase activity.
Firstly, we successfully designed and validated two genes, NS3-Hel and NS3-ZIKA, and obtained a plasmid pET28a from the laboratory. We then transformed pET28a into DH5a through enzymatic cleavage, enzyme-linked construction of the plasmid, and heat shock. The construction of target strains was successfully obtained per the verification of electrophoresis gel and sequencing.
After transferring the recombinant plasmid into BL21 to induce its expression by IPTG, we purified the two proteins to obtain two pure proteins by His-Tag which were validated by SDS-PAGE.
We then reacted the purified protein with ATP, MOPS Buffer, MgCl2, and double-chain substrate for 15 minutes and measured the fluorescence. The FRET result confirmed our proteins functioned well that possesses helicase activity, the fluorescence tended to decline as the protein concentration gradually increased.
Overall, we successfully obtained the engineered strains and NS3 proteins with good activity which means we made some progress in our research goal to develop a drug screening kit against the dengue virus.
Dengue is an acute febrile disease caused by mosquito-borne dengue viruses (DENVs), consisting of four serotypes (DENV 1 to 4). In our experiment, we only used one serotype. Thus, in the future, we could use more serotypes to improve our test kit. We can determine the optimal inhibitory concentration to optimize the conditions for sensitivity and specificity. We can maximize the engagement of different NS3 proteins and the helicase inhibitor compounds. By doing so, we can improve the effectiveness of our test kit. Additionally, we could try different combinations to verify our results and find a more efficient one. These optimizations will help us improve the accuracy and efficiency of our test kit, making it a more effective tool for diagnosing dengue.
[1]. World Health Organization: WHO & World Health Organization: WHO. (2023). Dengue and severe dengue. www.who.int. https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue.
[2]. Hussain S A .An Introduction to Fluorescence Resonance Energy Transfer (FRET).2009[2023-09-19].DOI:doi:http://dx.doi.org/.